Cationic lipid

ABSTRACT

The present invention provides a compound represented by the formula (Ia) as a novel cationic lipid that forms a lipid particle and also provides a lipid particle comprising the compound. The present invention further provides a nucleic acid lipid particle containing the lipid particle, and a pharmaceutical composition containing the nucleic acid lipid particle as an active ingredient.

This application is continuation of U.S. application Ser. No.14/903,711, filed Jan. 8, 2016, entitled “Cationic Lipid,” which is anational stage application under 35 U.S.C. § 371 of InternationalApplication No. PCT/JP2014/068002, filed Jul. 7, 2014, entitled “NovelLipid,” which claims priority to Japanese Patent Application No.2013-142677, filed Jul. 8, 2013.

TECHNICAL FIELD

The present invention relates to a novel cationic lipid, a novelcationic lipid that forms a lipid particle, a lipid particle comprisingthe cationic lipid, a nucleic acid lipid particle comprising the lipidparticle together with a nucleic acid, a pharmaceutical compositioncontaining the nucleic acid lipid particle as an active ingredient, anda treatment method using the pharmaceutical composition.

BACKGROUND ART

Methods for inhibiting the expression of a target gene in cells,tissues, or individuals include an approach in which double-stranded RNAis introduced into the cells, tissues, or individuals. By thisintroduction of double-stranded RNA, mRNA having homology to thesequence is degraded such that the expression of the target gene isinhibited. This effect is called “RNA interference” or “RNAi”. RNAinterference was originally reported in C. elegans (see e.g., Non PatentReference 1) and then also reported in plants (see e.g., Non PatentReference 2).

Double-stranded RNA consisting of 21-nucleotide sense and antisensestrands having a 2-nucleotide overhang at the 3′-end (small interferingRNA: siRNA) has been reported to have an RNA interference effect incultured cells of vertebrates (see e.g., Non Patent Reference 3). siRNAis considered to be useful for the identification of gene functions,screening of cell lines suitable for useful substance production,regulation of genes involved in disease, etc., but it ischaracteristically degraded easily by RNase (see e.g., Non PatentReference 4).

Since a double-stranded polynucleotide such as siRNA or modified siRNAis a molecule having a molecular weight on the order of 13,000, watersolubility, and electric charge, a delivery technique such as atransfection reagent is generally used for allowing the double-strandedpolynucleotide to permeate a cell membrane (see e.g., Non PatentReference 5). Particularly, liposomes are widely used in the delivery ofnucleic acid molecules by encapsulating a nucleic acid molecule such asplasmid DNA into a liposome to form a nucleic acid lipid particle (seee.g., Non Patent Reference 6). Also, a liposome containing a cationiclipid has been reported to be able to deliver siRNA into cells byforming a nucleic acid lipid particle through mixing with the siRNA (seee.g., Patent Reference 1). The cationic lipid, however, is anon-biological component. In this respect, a cationic lipid that can beused at a low concentration has been demanded. A dimethylaminovalericacid derivative (Patent Reference 1), a dimethylaminobutyric acidderivative (Patent Reference 2), a dimethylaminoethylcarbonatederivative (Patent Reference 3), or the like is known as the cationiclipid.

The present inventors have conducted diligent studies to obtain a lipidparticle consisting of a cationic lipid that can encapsulate therein anucleic acid such as a double-stranded polynucleotide (e.g., siRNA),DNA, or an antisense oligonucleotide and can be used at a lowconcentration. As a result, the present inventors have completed thepresent invention by finding a novel cationic lipid and further findinga nucleic acid lipid particle comprising the novel cationic lipid thatcan encapsulate therein a nucleic acid molecule, can be used at a lowconcentration, and permits a high level of delivery into cells.

CITATION LIST Patent Reference

Patent Reference 1: International Publication No. WO 2012/108397

Patent Reference 2: International Publication No. WO 2012/054365

Patent Reference 3: International Publication No. WO 2010/054405

Non Patent Reference

Non Patent Reference 1: Nature, 1998, Vol. 391, p. 806-811

Non Patent Reference 2: Science, 1999, Vol. 286, p. 950-952

Non Patent Reference 3: Nature, 2001, Vol. 411, p. 494-498

Non Patent Reference 4: Clinical Chemistry, 2002, Vol. 48, p. 1647-1653

Non Patent Reference 5: Journal of Medicinal Chemistry, 2010, Vol. 57,p. 7887-7901

Non Patent Reference 6: Gene Therapy 1999, Vol. 6, p. 271-281

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a novel cationic lipidthat forms a lipid particle.

Another object of the present invention is to provide a novel cationiclipid that forms a lipid particle in combination with an amphipathiclipid, a sterol, and a lipid reducing aggregation during lipid particleformation.

A further object of the present invention is to provide a lipid particlecomprising the cationic lipid.

A further object of the present invention is to provide a nucleic acidlipid particle comprising the lipid particle and further a nucleic acid.

A further object of the present invention is to provide a pharmaceuticalcomposition containing the nucleic acid lipid particle as an activeingredient.

A further object of the present invention is to provide a treatmentmethod using the pharmaceutical composition.

Solution to Problem

Specifically, the present invention provides:

(1) A cationic lipid represented by the general formula (Ia) or apharmacologically acceptable salt thereof:

whereinR¹ and R² each independently represent a hydrogen atom, a C₁-C₆ alkylgroup optionally having one or more substituents selected fromsubstituent group α, a C₂-C₆ alkenyl group optionally having one or moresubstituents selected from substituent group α, a C₂-C₆ alkynyl groupoptionally having one or more substituents selected from substituentgroup α, or a C₃-C₇ cycloalkyl group optionally having one or moresubstituents selected from substituent group α, or R¹ and R² form a 3-to 10-membered heterocyclic ring together with the nitrogen atom bondedthereto, wherein the heterocyclic ring optionally has one or moresubstituents selected from substituent group α and optionally containsone or more atoms selected from a nitrogen atom, an oxygen atom, and asulfur atom, in addition to the nitrogen atom bonded to R¹ and R², asatoms constituting the heterocyclic ring;R⁸ represents a hydrogen atom or a C₁-C₆ alkyl group optionally havingone or more substituents selected from substituent group α;or R¹ and R⁸ together represent a group —(CH₂)_(q)—; substituent group αrepresents the group consisting of a halogen atom, an oxo group, ahydroxy group, a sulfanyl group, an amino group, a cyano group, a C₁-C₆alkyl group, a C₁-C₆ halogenated alkyl group, a C₁-C₆ alkoxy group, aC₁-C₆ alkylsulfanyl group, a C₁-C₆ alkylamino group, and a C₁-C₇alkanoyl group;L¹ represents a C₁₀-C₂₄ alkyl group optionally having one or moresubstituents selected from substituent group β1, a C₁₀-C₂₄ alkenyl groupoptionally having one or more substituents selected from substituentgroup β1, a C₃-C₂₄ alkynyl group optionally having one or moresubstituents selected from substituent group β1, or a (C₁-C₁₀alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) group optionally having one or moresubstituents selected from substituent group β1;L² represents, independently of L¹, a C₁₀-C₂₄ alkyl group optionallyhaving one or more substituents selected from substituent group β1, aC₁₀-C₂₄ alkenyl group optionally having one or more substituentsselected from substituent group β1, a C₃-C₂₄ alkynyl group optionallyhaving one or more substituents selected from substituent group β1, a(C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) group optionally having one ormore substituents selected from substituent group β1, a (C₁₀-C₂₄alkoxy)methyl group optionally having one or more substituents selectedfrom substituent group β1, a (C₁₀-C₂₄ alkenyl)oxymethyl group optionallyhaving one or more substituents selected from substituent group β1, a(C₃-C₂₄ alkynyl)oxymethyl group optionally having one or moresubstituents selected from substituent group β1, or a (C₁-C₁₀alkyl)-(Q)_(k)-(C₁-C₁₀ alkoxy)methyl group optionally having one or moresubstituents selected from substituent group β1;substituent group β1 represents the group consisting of a halogen atom,an oxo group, a cyano group, a C₁-C₆ alkyl group, a C₁-C₆ halogenatedalkyl group, a C₁-C₆ alkoxy group, a C₁-C₆ alkylsulfanyl group, a C₁-C₇alkanoyl group, a C₁-C₇ alkanoyloxy group, a C₃-C₇ alkoxyalkoxy group, a(C₁-C₆ alkoxy)carbonyl group, a (C₁-C₆ alkoxy)carboxyl group, a (C₁-C₆alkoxy)carbamoyl group, and a (C₁-C₆ alkylamino)carboxyl group;Q represents a group represented by the following formula (II):

when L¹ and L² each have one or more substituents selected fromsubstituent group β1 and substituent group β1 is a C₁-C₆ alkyl group, aC₁-C₆ alkoxy group, a C₁-C₆ alkylsulfanyl group, a C₁-C₇ alkanoyl group,or a C₁-C₇ alkanoyloxy group, the substituent(s) selected fromsubstituent group β1 in L¹ and the substituent(s) selected fromsubstituent group β1 in L² optionally bind to each other to form acyclic structure;k represents 1, 2, 3, 4, 5, 6, or 7;m represents 0 or 1;p represents 0, 1, or 2;q represents 1, 2, 3, or 4; andr represents 0, 1, 2, or 3,provided that p+r is 2 or larger, or q+r is 2 or larger;

(2) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R² are each independently aC₁-C₆ alkyl group optionally having one or more substituents selectedfrom substituent group α;

(3) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R² are each independently aC₁-C₃ alkyl group;

(4) The cationic lipid according to claim 2 (1) or a pharmacologicallyacceptable salt thereof, wherein both R¹ and R² are methyl groups;

(5) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R² form azetidine, pyrrolidine,piperidine, azepane, dihydropyrrole, dihydropyridine,tetrahydropyridine, piperazine, morpholine, dihydrooxazole, ordihydrothiazole optionally having one or more substituents selected fromsubstituent group α, together with the nitrogen atom bonded thereto;

(6) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R² form azetidine, pyrrolidine,piperidine, or morpholine optionally having one or more substituentsselected from substituent group α, together with the nitrogen atombonded thereto;

(7) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R² form azetidine, pyrrolidine,or morpholine together with the nitrogen atom bonded thereto;

(8) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R¹ and R⁸ together represent a group—(CH₂)_(q)—; p+q is 2, 3, or 4; and R² is a C₁-C₃ alkyl group optionallyhaving one or more substituents selected from substituent group α;

(9) The cationic lipid according to (8) or a pharmacologicallyacceptable salt thereof, wherein R² is a C₁-C₃ alkyl group;

(10) The cationic lipid according to (8) or a pharmacologicallyacceptable salt thereof, wherein R² is a methyl group;

(11) The cationic lipid according to any one of (1) to (10) or apharmacologically acceptable salt thereof, wherein L¹ is a C₁₇-C₁₉ alkylgroup optionally having one or more substituents selected fromsubstituent group β1, a C₁₇-C₁₉ alkenyl group optionally having one ormore substituents selected from substituent group β1, or a (C₁-C₄alkyl)-(Q)_(k)-(C₄-C₉ alkyl) group optionally having one or moresubstituents selected from substituent group β1; and k is 1, 2, or 3;

(12) The cationic lipid according to any one of (1) to (10) or apharmacologically acceptable salt thereof, wherein L¹ is a heptadecenylgroup, an octadecenyl group, a nonadecenyl group, a heptadecadienylgroup, an octadecadienyl group, a nonadecadienyl group, aheptadecatrienyl group, an octadecatrienyl group, or a nonadecatrienylgroup optionally having one or more substituents selected fromsubstituent group β1;

(13) The cationic lipid according to any one of (1) to (10) or apharmacologically acceptable salt thereof, wherein L¹ is a(R)-11-acetyloxy-cis-8-heptadecenyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyl group, acis-9-octadecenyl group (oleyl group), a cis-8,11-heptadecadienyl group,a cis-9,12-octadecadienyl group (linoleyl group), acis-10,13-nonadecadienyl group, or a cis-6,9,12-octadecatrienyl group(linolenyl group);

(14) The cationic lipid according to any one of (1) to (13) or apharmacologically acceptable salt thereof, wherein L² is a C₁₀-C₁₉ alkylgroup optionally having one or more substituents selected fromsubstituent group β1, a C₁₀-C₁₉ alkenyl group optionally having one ormore substituents selected from substituent group β1, a (C₁-C₄alkyl)-(Q)_(k)-(C₄-C₉ alkyl) group optionally having one or moresubstituents selected from substituent group β1, a (C₁₀-C₁₉alkoxy)methyl group optionally having one or more substituents selectedfrom substituent group β1, a (C₁₀-C₁₉ alkenyl)oxymethyl group optionallyhaving one or more substituents selected from substituent group β1, or a(C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkoxy)methyl group optionally having oneor more substituents selected from substituent group β1; and k is 1, 2,or 3;

(15) The cationic lipid according to any one of (1) to (13) or apharmacologically acceptable salt thereof, wherein L² is a decyl group,a decenyl group, an undecyl group, an undecenyl group, a dodecyl group,a dodecenyl group, a decadienyl group, an undecadienyl group, adodecadienyl group, a heptadecadienyl group, an octadecadienyl group, anonadecadienyl group, a heptadecatrienyl group, an octadecatrienylgroup, a nonadecatrienyl group, a decyloxymethyl group, adecenyloxymethyl group, an undecyloxymethyl group, an undecenyloxymethylgroup, a dodecyloxymethyl group, a dodecenyloxymethyl group, adecadienyloxymethyl group, an undecadienyloxymethyl group, adodecadienyloxymethyl group, a heptadecadienyloxymethyl group, anoctadecadienyloxymethyl group, a nonadecadienyloxymethyl group, aheptadecatrienyloxymethyl group, an octadecatrienyloxymethyl group, or anonadecatrienyloxymethyl group optionally having one or moresubstituents selected from substituent group β1;

(16) The cationic lipid according to any one of (1) to (13) or apharmacologically acceptable salt thereof, wherein L² is a decyl group,a cis-7-decenyl group, a (R)-11-acetyloxy-cis-8-heptadecenyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyl group, acis-9-octadecenyl group (oleyl group), a cis-8,11-heptadecadienyl group,a cis-9,12-octadecadienyl group (linoleyl group), acis-10,13-nonadecadienyl group, a cis-6,9,12-octadecatrienyl group(linolenyl group), a decyloxymethyl group, a cis-7-decenyloxymethylgroup, a (R)-11-acetyloxy-cis-8-heptadecenyloxymethyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyloxymethyl group,a cis-9-octadecenyloxymethyl group (oleyloxymethyl group), acis-8,11-heptadecadienyloxymethyl group, acis-9,12-octadecadienyloxymethyl group (linoleyloxymethyl group), acis-10,13-nonadecadienyloxymethyl group, or acis-6,9,12-octadecatrienyloxymethyl group (linolenyloxymethyl group);

(17) The cationic lipid according to any one of (1) to (16) or apharmacologically acceptable salt thereof, wherein m is 0;

(18) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein both R¹ and R² are methyl groups; R⁸ isa hydrogen atom; L¹ is a C₁₇-C₁₉ alkyl group optionally substituted byone acetyloxy group, or a C₁₇-C₁₉ alkenyl group optionally substitutedby one acetyloxy group; L² is a C₁₀-C₁₉ alkyl group optionallysubstituted by one acetyloxy group, a C₁₀-C₁₉ alkenyl group optionallysubstituted by one acetyloxy group, a (C₁₀-C₁₉ alkoxy)methyl groupoptionally substituted by one acetyloxy group, or a (C₁₀-C₁₉alkenyl)oxymethyl group optionally substituted by one acetyloxy group;p+r is 2; and m is 0;

(19) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R² is a methyl group; R¹ and R⁸together represent a group —(CH₂)_(q)—; L¹ is a C₁₇-C₁₉ alkyl groupoptionally substituted by one acetyloxy group, or a C₁₇-C₁₉ alkenylgroup optionally substituted by one acetyloxy group; L² is a C₁₀-C₁₉alkyl group optionally substituted by one acetyloxy group, a C₁₀-C₁₉alkenyl group optionally substituted by one acetyloxy group, a (C₁₀-C₁₉alkoxy)methyl group optionally substituted by one acetyloxy group, or a(C₁₀-C₁₉ alkenyl)oxymethyl group optionally substituted by one acetyloxygroup; p is 2; q is 2; r is 0; and m is 0;

(20) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R² is a methyl group; R¹ and R⁸together represent a group —(CH₂)_(q)—; L¹ is a C₁₇-C₁₉ alkyl groupoptionally substituted by one acetyloxy group, or a C₁₇-C₁₉ alkenylgroup optionally substituted by one acetyloxy group; L² is a C₁₀-C₁₉alkyl group optionally substituted by one acetyloxy group, a C₁₀-C₁₉alkenyl group optionally substituted by one acetyloxy group, a (C₁₀-C₁₉alkoxy)methyl group optionally substituted by one acetyloxy group, or a(C₁₀-C₁₉ alkenyl)oxymethyl group optionally substituted by one acetyloxygroup; p is 1; q is 2 or 3; r is 1; and m is 0;

(21) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein R² is a methyl group; R¹ and R⁸together represent a group —(CH₂)_(q)—; L¹ is a C₁₇-C₁₉ alkyl groupoptionally substituted by one acetyloxy group, or a C₁₇-C₁₉ alkenylgroup optionally substituted by one acetyloxy group; L² is a C₁₀-C₁₉alkyl group optionally substituted by one acetyloxy group, a C₁₀-C₁₉alkenyl group optionally substituted by one acetyloxy group, a (C₁₀-C₁₉alkoxy)methyl group optionally substituted by one acetyloxy group, or a(C₁₀-C₁₉ alkenyl)oxymethyl group optionally substituted by one acetyloxygroup; p is 0; q is 3 or 4; r is 2; and m is 0;

(22) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(23) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(24) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(25) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(26) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(27) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(28) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(29) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(30) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(31) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(32) The cationic lipid according to (1) or a pharmacologicallyacceptable salt thereof, wherein the cationic lipid is represented bythe formula:

(33) A lipid particle comprising at least a cationic lipid according toany one of (1) to (32);

(34) The lipid particle according to (33), further comprising a lipidreducing aggregation during lipid particle formation;

(35) The lipid particle according to (34), wherein the lipid reducingaggregation during lipid particle formation is a PEG-lipid;

(36) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA), or1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol;

(37) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]-carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA);

(38) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dipalmityloxypropyl-3-amine (PEG-C-DPA), or1,2-dipalmitoyl-sn-glycerol methoxypolyethylene glycol;

(39) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dipalmityloxypropyl-3-amine (PEG-C-DPA);

(40) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearyloxypropyl-3-amine (PEG-C-DSA), or1,2-distearoyl-sn-glycerol methoxypolyethylene glycol;

(41) The lipid particle according to (35), wherein the PEG-lipid isN-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearyloxypropyl-3-amine (PEG-C-DSA);

(42) The lipid particle according to any one of (35) to (41), whereinthe PEG has a molecular weight of 1,000 to 5,000;

(43) The lipid particle according to any one of (35) to (41), whereinthe PEG has a molecular weight of 1,800 to 2,200;

(44) The lipid particle according to any one of (33) to (43), furthercomprising a sterol;

(45) The lipid particle according to (44), wherein the sterol ischolesterol;

(46) The lipid particle according to any one of claims 33) to (45),further comprising an amphipathic lipid;

(47) The lipid particle according to (46), wherein the amphipathic lipidis at least any one selected from distearoylphosphatidylcholine (DSPC),dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine(DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC),dioleoylphosphatidylethanolamine (DOPE), and sphingomyelin (SM);

(48) The lipid particle according to (46), wherein the amphipathic lipidis distearoylphosphatidylcholine (DSPC) ordipalmitoylphosphatidylcholine (DPPC);

(49) The lipid particle according to any one of (46) to (48), whereinthe lipid composition of the amphipathic lipid, the sterol, the cationiclipid, and the lipid reducing aggregation during lipid particleformation is 25% or less of the amphipathic lipid, 15% or more of thesterol, 20% to 70% of the cationic lipid, and 1% to 10% of the lipidreducing aggregation during lipid particle formation, in terms of molarquantity;

(50) The lipid particle according to any one of (46) to (48), whereinthe lipid composition of the amphipathic lipid, the sterol, the cationiclipid, and the lipid reducing aggregation during lipid particleformation is 15% or less of the amphipathic lipid, 32% or more of thesterol, 45% to 65% of the cationic lipid, and 1.5% to 3% of the lipidreducing aggregation during lipid particle formation, in terms of molarquantity;

(51) A nucleic acid lipid particle comprising a lipid particle accordingto any one of (33) to (50) and a nucleic acid;

(52) The nucleic acid lipid particle according to (51), wherein thenucleic acid is any one selected from the group consisting of asingle-stranded DNA, a single-stranded RNA, a single-strandedpolynucleotide of a DNA and an RNA mixed with each other, adouble-stranded DNA, a double-stranded RNA, a DNA-RNA hybridpolynucleotide, and two polynucleotides of a DNA and an RNA mixed witheach other;

(53) The nucleic acid lipid particle according to (51), wherein thenucleic acid is a single-stranded or double-stranded polynucleotidehaving an RNA interference effect;

(54) The nucleic acid lipid particle according to (51), wherein thenucleic acid is a single-stranded RNA;

(55) The nucleic acid lipid particle according to any one of (51) to(54), wherein the ratio of the number of molecules of the cationic lipid(N) to the number of phosphorus atoms derived from the nucleic acid (P)is 2.0 to 9.0;

(56) The nucleic acid lipid particle according to any one of (51) to(54), wherein the ratio of the number of molecules of the cationic lipid(N) to the number of phosphorus atoms derived from the nucleic acid (P)is 3.0 to 9.0;

(57) The nucleic acid lipid particle according to any one of (51) to(56), wherein the average particle size is approximately 30 nm toapproximately 300 nm;

(58) The nucleic acid lipid particle according to any one of (51) to(56), wherein the average particle size is approximately 30 nm toapproximately 200 nm;

(59) The nucleic acid lipid particle according to any one of (51) to(56), wherein the average particle size is approximately 30 nm toapproximately 100 nm;

(60) A pharmaceutical composition comprising a nucleic acid lipidparticle according to any one of (51) to (59) as an active ingredient;

(61) The pharmaceutical composition according to (60), wherein thepharmaceutical composition is intended for the treatment or preventionof a disease derived from the expression of a target gene;

(62) The pharmaceutical composition according to (60), wherein thedisease derived from the expression of a target gene is cancer, liverdisease, gallbladder disease, fibrosis, anemia, or genetic disease;

(63) A method for inhibiting the expression of a target gene, comprisingadministering a nucleic acid lipid particle according to any one of (51)to (59) to a mammal;

(64) A method for treating or preventing a disease derived from theexpression of a target gene, comprising administering a nucleic acidlipid particle according to any one of (51) to (59) to a mammal;

(65) The method according to (64), wherein the disease derived from theexpression of a target gene is cancer; and

(66) A cationic lipid represented by the general formula (I):

whereinR¹ and R² each independently represent a hydrogen atom, a C₁-C₆ alkylgroup optionally having one or more substituents selected fromsubstituent group α, a C₂-C₆ alkenyl group optionally having one or moresubstituents selected from substituent group α, a C₂-C₆ alkynyl groupoptionally having one or more substituents selected from substituentgroup α, or a C₃-C₇ cycloalkyl group optionally having one or moresubstituents selected from substituent group α, or R¹ and R² form a 3-to 10-membered heterocyclic ring together with the nitrogen atom bondedthereto, wherein the heterocyclic ring optionally has one or moresubstituents selected from substituent group α and optionally containsone or more atoms selected from a nitrogen atom, an oxygen atom, and asulfur atom, in addition to the nitrogen atom bonded to R¹ and R², asatoms constituting the heterocyclic ring;R³ and R⁴ each independently represent a hydrogen atom or a C₁-C₆ alkylgroup optionally having one or more substituents selected fromsubstituent group α, or R³ and R⁴ form a 3- to 10-membered hydrocarbonring together with the carbon atom bonded thereto;or R¹ forms a 3- to 10-membered heterocyclic ring together with thenitrogen atom bonded to R¹, R³, and the carbon atom bonded to R³,wherein the heterocyclic ring optionally has one or more substituentsselected from substituent group α and optionally contains one or moreatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom,in addition to the nitrogen atom bonded to R¹, as atoms constituting theheterocyclic ring;R² and R⁴ each independently represent a hydrogen atom, a C₁-C₆ alkylgroup optionally having one or more substituents selected fromsubstituent group α, a C₂-C₆ alkenyl group optionally having one or moresubstituents selected from substituent group α, or a C₂-C₆ alkynyl groupoptionally having one or more substituents selected from substituentgroup α;R⁵ and R⁶ each independently represent a hydrogen atom or a C₁-C₃ alkylgroup;R⁷ represents a hydrogen atom or a C₁-C₆ alkyl group optionally havingone or more substituents selected from substituent group α;substituent group α represents the group consisting of a halogen atom,an oxo group, a hydroxy group, a sulfanyl group, an amino group, a cyanogroup, a C₁-C₆ alkyl group, a C₁-C₆ halogenated alkyl group, a C₁-C₆alkoxy group, a C₁-C₆ alkylsulfanyl group, a C₁-C₆ alkylamino group, anda C₁-C₇ alkanoyl group;L¹ and L² each independently represent a C₁₀-C₂₄ alkyl group optionallyhaving one or more substituents selected from substituent group β, aC₁₀-C₂₄ alkenyl group optionally having one or more substituentsselected from substituent group β, a C₃-C₂₄ alkynyl group optionallyhaving one or more substituents selected from substituent group β, or a(C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) group optionally having one ormore substituents selected from substituent group β;substituent group β represents the group consisting of a halogen atom,an oxo group, a hydroxy group, a sulfanyl group, an amino group, a cyanogroup, a C₁-C₆ alkyl group, a C₁-C₆ halogenated alkyl group, a C₁-C₆alkoxy group, a C₁-C₆ alkylsulfanyl group, a C₁-C₇ alkanoyl group, aC₁-C₇ alkanoyloxy group, a C₃-C₇ alkoxyalkoxy group, a C₁-C₆alkoxycarbonyl group, a C₁-C₆ alkoxycarboxyl group, a C₁-C₆alkoxycarbamoyl group, and a C₁-C₆ alkylaminocarboxyl group;Q represents a group represented by the following formula (II):

when L¹ and L² each have one or more substituents selected fromsubstituent group β and substituent group β is a sulfanyl group, a C₁-C₆alkyl group, a C₁-C₆ alkoxy group, a C₁-C₆ alkylsulfanyl group, a C₁-C₇alkanoyl group, or a C₁-C₇ alkanoyloxy group, the substituent(s)selected from substituent group β in L¹ and the substituent(s) selectedfrom substituent group β in L² optionally bind to each other to form acyclic structure;k represents an integer of 1 to 7;m represents an integer of 0 or 1; andn represents an integer of 3 to 6.

Advantageous Effects of Invention

The present invention may provide a novel cationic lipid that forms alipid particle.

The present invention may also provide a novel cationic lipid that formsa lipid particle in combination with an amphipathic lipid, a sterol, anda lipid reducing aggregation during lipid particle formation.

The present invention may further provide a lipid particle comprisingthe cationic lipid.

The present invention may further provide a nucleic acid lipid particlecomprising the lipid particle and further a nucleic acid.

The present invention may further provide a pharmaceutical compositioncontaining the nucleic acid lipid particle as an active ingredient.

The present invention may further provide a method for treating adisease using the pharmaceutical composition.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram summarizing a method for producing an intermediate(A5) for use in the synthesis of a cationic lipid represented by theformula (I).

FIG. 2 is a diagram summarizing method B for use in the synthesis of thecationic lipid represented by the formula (I).

FIG. 3 is a diagram summarizing method C for use in the synthesis of thecationic lipid represented by the formula (I).

FIG. 4 is a diagram showing the structure of each nucleic acid having adouble-stranded structure among nucleic acids constituting nucleic acidlipid particles. In the diagram, the upper sequences represent sensestrands, and the lower sequences represent antisense strands. Forsymbols, the open square (□) represents an RNA, the filled circle (●)represents a DNA, and the open circle (◯) represents a 2′-O-methyl RNA.The line between the symbols represents a phosphodiester bond betweenthe nucleosides. In the diagram, p represents —P(═O)(OH)—. When p isbound, a hydrogen atom in the terminal hydroxy group of thepolynucleotide is removed. When the end of the polynucleotide isunbound, the 3′-end or 5′-end of the RNA, the DNA, or the 2′-O-methylRNA is an OH group. X represents a compound modifying the 5′-end of anantisense strand described in the paragraph “3-4-2. Modifieddouble-stranded polynucleotide” in the specification. “linker” means apolynucleotide linker described in the paragraph “3-4-3. Modifiedsingle-stranded polynucleotide” in the specification.

FIG. 5 is a diagram showing PLK-1 expression inhibitory activityexhibited, in tumor, by a nucleic acid lipid particle having a compoundof Example 19, 45, or 54 in Test Example 10.

FIG. 6 is a diagram showing that a nucleic acid lipid particlecontaining a compound of Example 8 in Test Example 11 promotes theexpression of mRNA. The upper boxes depict images of nuclei stained withHoechst, and the lower boxes depict images of mCherry.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the embodiments of the present invention will be describedin detail.

1. Cationic Lipid

The cationic lipid disclosed in the present specification can be usedalone and can be used in combination with an additional substance. Forexample, the cationic lipid can be used as a component constituting alipid particle and can be used as a component constituting a nucleicacid lipid particle.

1-1. Definition of Group

In the present invention, the “cationic lipid” is a lipid, somemolecules of which have a net positive charge according to pKa of thelipid at a selected pH such as physiological pH. The cationic lipid ofthe present invention is a lipid that can be ionized (ionizable lipid)and differs from a cationic lipid having quaternary amine, which is alipid, all molecules of which have a net positive charge at any pH(e.g., N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)).

The “C₁-C₆ alkyl group” in the definitions of R¹, R², R³, R⁴, R⁷, R⁸,substituent group α, and substituent group β refers to a linear orbranched alkyl group having 1 to 6 carbon atoms. Examples thereof caninclude a methyl group, an ethyl group, a n-propyl group, an isopropylgroup, a n-butyl group, a sec-butyl group, a tert-butyl group, an-pentyl group, an isopentyl group, a 2-methylbutyl group, a neopentylgroup, a 1-ethylpropyl group, a n-hexyl group, a 4-methylpentyl group, a3-methylpentyl group, a 2-methylpentyl group, a 1-methylpentyl group, a3,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 1,1-dimethylbutylgroup, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a2,3-dimethylbutyl group, and a 2-ethylbutyl group. The C₁-C₆ alkyl groupis preferably a C₁-C₄ alkyl group, more preferably a C₁-C₃ alkyl group.

The “C₁-C₃ alkyl group” in the definitions of R¹, R², R³, R⁵, and R⁶refers to a linear or branched alkyl group having 1 to 3 carbon atoms.Examples thereof can include a methyl group, an ethyl group, a propylgroup, and an isopropyl group. The C₁-C₃ alkyl group is preferably amethyl group.

The “C₂-C₆ alkenyl group” in the definitions of R¹, R², and R⁴ refers toa linear or branched alkenyl group having 2 to 6 carbon atoms. Examplesthereof can include a vinyl group, a 1-propenyl group, a 2-propenylgroup, an isopropenyl group, a 1-methyl-2-propenyl group, a2-methyl-2-propenyl group, a 1-butenyl group, a 2-butenyl group, a3-butenyl group, a 2-methyl-1-propenyl group, a 1-pentenyl group, a4-pentenyl group, a 1-methyl-4-pentenyl group, and a 5-hexenyl group.

The “C₂-C₆ alkynyl group” in the definitions of R¹, R², and R⁴ refers toa linear or branched alkynyl group having 2 to 6 carbon atoms. Examplesthereof can include an ethynyl group, a 1-propynyl group, a 2-propynylgroup, a 1-methyl-2-propynyl group, a 1-butynyl group, a 2-butynylgroup, a 3-butynyl group, a 1-pentynyl group, a 4-pentynyl group, a1-methyl-4-pentynyl group, and a 5-hexynyl group.

The “C₃-C₇ cycloalkyl group” in the definitions of R¹ and R² refers to acycloalkyl group having 3 to 7 carbon atoms. Examples thereof caninclude a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, acyclohexyl group, and a cycloheptyl group.

The “3- to 10-membered heterocyclic ring” in the definitions of R¹, R²,and R³ refers to a saturated or partially unsaturated 3- to 10-memberedmonocyclic or bicyclic heterocyclic group containing at least onenitrogen atom and optionally further containing one or more atomsselected from the group consisting of a nitrogen atom, a sulfur atom,and an oxygen atom. Examples thereof can include azetidine, pyrrolidine,piperidine, azepane, dihydropyrrole, dihydropyridine,tetrahydropyridine, piperazine, morpholine, dihydrooxazole, anddihydrothiazole. The heterocyclic ring formed by R¹ and R² together withthe nitrogen atom bonded thereto is preferably azetidine, pyrrolidine,or morpholine. The heterocyclic ring formed by R¹ together with thenitrogen atom bonded to R¹, R³, and the carbon atom bonded to R³ ispreferably azetidine, pyrrolidine, piperidine, or morpholine.

The “3- to 10-membered hydrocarbon ring” in the definitions of R³ and R⁴refers to a saturated hydrocarbon ring group having 3 to 10 carbonatoms. Examples thereof can include a cyclopropyl group, a cyclobutylgroup, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, acyclooctyl group, a cyclononyl group, and a cyclodecanyl group.

The “halogen atom” in the definitions of substituent group α andsubstituent group β is a fluorine atom, a chlorine atom, a bromine atom,or an iodine atom and is preferably a fluorine atom.

The “C₁-C₆ halogenated alkyl group” in the definitions of substituentgroup α and substituent group β refers to a group in which one or twohydrogen atoms in the “C₁-C₆ alkyl group” described above are replacedwith the “halogen atom” described above. Examples thereof can include afluoromethyl group, a chloromethyl group, a 1-fluoroethyl group, a1-chloroethyl group, a 2-fluoroethyl group, and a 1,2-difluoropropylgroup. The C₁-C₆ halogenated alkyl group is preferably a C₁-C₄halogenated alkyl group, more preferably a C₁-C₃ halogenated alkylgroup.

The “C₁-C₆ alkoxy group” in the definitions of substituent group α andsubstituent group β refers to a group in which the “C₁-C₆ alkyl group”described above is bonded to an oxygen atom. Examples thereof caninclude a methoxy group, an ethoxy group, a n-propoxy group, a n-butoxygroup, a s-butoxy group, a tert-butoxy group, and a n-pentoxy group. TheC₁-C₆ alkoxy group is preferably a C₁-C₄ alkoxy group, more preferably aC₁-C₂ alkoxy group.

The “C₁-C₆ alkylsulfanyl group” in the definitions of substituent groupα and substituent group β refers to a group in which the “C₁-C₆ alkylgroup” described above is bonded to a sulfur atom. Examples thereof caninclude a methylsulfanyl group, an ethylsulfanyl group, an-propylsulfanyl group, a n-butylsulfanyl group, a s-butylsulfanylgroup, a tert-butylsulfanyl group, and a n-pentylsulfanyl group. TheC₁-C₆ alkylsulfanyl group is preferably a C₁-C₄ alkylsulfanyl group,more preferably a C₁-C₂ alkylsulfanyl group.

The “C₁-C₆ alkylamino group” in the definition of substituent group αrefers to a group in which the “C₁-C₆ alkyl group” described above isbonded to a nitrogen atom. Examples thereof can include a methylaminogroup, an ethylamino group, a n-propylamino group, a n-butylamino group,a s-butylamino group, a tert-butylamino group, a n-pentylamino group, an-hexylamino group, a N,N-dimethylamino group, a N,N-diethylamino group,a N,N-di-n-propylamino group, a N,N-diisopropylamino group, aN,N-di-n-butylamino group, a N,N-diisobutylamino group, aN,N-di-s-butylamino group, and a N,N-di-tert-butylamino group. The C₁-C₆alkylamino group is preferably a C₁-C₄ alkylamino group, more preferablya C₁-C₂ alkylamino group.

The “C₁-C₇ alkanoyl group” in the definitions of substituent group α andsubstituent group β refers to an alkanoyl group having 1 to 7 carbonatoms. Examples thereof can include a formyl group, an acetyl group, apropionyl group, a butyryl group, an isobutyryl group, a pentanoylgroup, a pivaloyl group, a valeryl group, an isovaleryl group, ahexanoyl group, and a heptanoyl group.

The “C₁-C₇ alkanoyloxy group” in the definition of substituent group βrefers to a group in which the “C₁-C₇ alkanoyl group” described above isbonded to an oxygen atom. Examples thereof can include a formyloxygroup, an acetyloxy group, a propionyloxy group, a butyryloxy group, anisobutyryloxy group, a pentanoyloxy group, a pivaloyloxy group, avaleryloxy group, an isovaleryloxy group, a hexanoyloxy group, and aheptanoyloxy group.

The “C₃-C₇ alkoxyalkoxy group” in the definition of substituent group βrefers to a group in which one or two carbon atoms of a linear,branched, or cyclic alkane having 3 to 7 carbon atoms are replaced withan oxygen atom and the resulting group is further bonded to an oxygenatom (except for peroxide). Examples thereof can include amethoxymethoxy group, an ethoxymethoxy group, an ethoxyethoxy group, a2-tetrahydrofuranyloxy group, and a 2-tetrahydropyranyloxy group.

The “(C₁-C₆ alkoxy)carbonyl group” in the definition of substituentgroup β refers to a group in which the “C₁-C₆ alkoxy group” describedabove is bonded to a carbonyl group. Examples thereof can include amethoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonyl group,an isopropoxycarbonyl group, a butoxycarbonyl group, anisobutoxycarbonyl group, a sec-butoxycarbonyl group, atert-butoxycarbonyl group, a pentyloxycarbonyl group, and ahexyloxycarbonyl group.

The “(C₁-C₆ alkoxy)carboxyl group” in the definition of substituentgroup β refers to a group in which the “C₁-C₆ alkoxy group” describedabove is bonded to a carboxyl group. Examples thereof can include amethoxycarboxyl group, an ethoxycarboxyl group, a propoxycarboxyl group,an isopropoxycarboxyl group, a butoxycarboxyl group, anisobutoxycarboxyl group, a sec-butoxycarboxyl group, atert-butoxycarboxyl group, a pentyloxycarboxyl group, and ahexyloxycarboxyl group.

The “(C₁-C₆ alkoxy)carbamoyl group” in the definition of substituentgroup β refers to a group in which the “C₁-C₆ alkoxy group” describedabove is bonded to a carbamoyl group. Examples thereof can include amethoxycarbamoyl group, an ethoxycarbamoyl group, a propoxycarbamoylgroup, an isopropoxycarbamoyl group, a butoxycarbamoyl group, anisobutoxycarbamoyl group, a sec-butoxycarbamoyl group, atert-butoxycarbamoyl group, a pentyloxycarbamoyl group, and ahexyloxycarbamoyl group.

The “(C₁-C₆ alkylamino)carboxyl group” in the definition of substituentgroup β refers to a group in which the “C₁-C₆ alkylamino group”described above is bonded to a carboxyl group. Examples thereof caninclude a methylaminocarboxyl group, an ethylaminocarboxyl group, an-propylaminocarboxyl group, a n-butylaminocarboxyl group, as-butylaminocarboxyl group, a tert-butylaminocarboxyl group, an-pentylaminocarboxyl group, a n-hexylaminocarboxyl group, aN,N-dimethylaminocarboxyl group, a N,N-diethylaminocarboxyl group, aN,N-di-n-propylaminocarboxyl group, a N,N-diisopropylaminocarboxylgroup, a N,N-di-n-butylaminocarboxyl group, aN,N-diisobutylaminocarboxyl group, a N,N-di-s-butylaminocarboxyl group,and a N,N-di-tert-butylaminocarboxyl group.

The “C₁₀-C₂₄ alkyl group” in the definitions of L¹ and L² refer to alinear alkyl group having 10 to 24 carbon atoms. Examples thereof caninclude a decyl group, an undecyl group, a dodecyl group, a tridecylgroup, a tetradecyl group, a pentadecyl group, a hexadecyl group, aheptadecyl group, an octadecyl group, a nonadecyl group, an icosylgroup, a henicosyl group, a docosyl group, a tricosyl group, and atetracosyl group. The “C₁₀-C₂₄ alkyl group” represented by L¹ ispreferably a heptadecyl group, an octadecyl group, or a nonadecyl group.The “C₁₀-C₂₄ alkyl group” represented by L² is preferably a decyl group,an undecyl group, or a dodecyl group.

The “C₁₀-C₂₄ alkenyl group” in the definitions of L¹ and L² refers to alinear alkenyl group having 10 to 24 carbon atoms. The “C₁₀-C₂₄ alkenylgroup” in the present application includes any of a C₁₀-C₂₄ alkadienylgroup, a C₁₀-C₂₄ alkatrienyl group, and a C₁₀-C₂₄ alkatetraenyl group.Examples thereof can include a decenyl group, an undecenyl group, adodecenyl group, a tridecenyl group, a tetradecenyl group, apentadecenyl group, a hexadecenyl group, a heptadecenyl group, anoctadecenyl group, a nonadecenyl group, an icosenyl group, a henicosenylgroup, a docosenyl group, a tricosenyl group, a tetracosenyl group, adecadienyl group, an undecadienyl group, a dodecadienyl group, atridecadienyl group, a tetradecadienyl group, a pentadecadienyl group, ahexadecadienyl group, a heptadecadienyl group, an octadecadienyl group,a nonadecadienyl group, an icosadienyl group, a henicosadienyl group, adocosadienyl group, a tricosadienyl group, a tetracosadienyl group, adecatrienyl group, an undecatrienyl group, a dodecatrienyl group, atridecatrienyl group, a tetradecatrienyl group, a pentadecatrienylgroup, a hexadecatrienyl group, a heptadecatrienyl group, anoctadecatrienyl group, a nonadecatrienyl group, an icosatrienyl group, ahenicosatrienyl group, a docosatrienyl group, a tricosatrienyl group,and a tetracosatrienyl group. The “C₁₀-C₂₄ alkenyl group” represented byL¹ is preferably a heptadecenyl group, an octadecenyl group, anonadecenyl group, a heptadecadienyl group, an octadecadienyl group, anonadecadienyl group, a heptadecatrienyl group, an octadecatrienylgroup, or a nonadecatrienyl group. L¹ is preferably a(R)-11-acetyloxy-cis-8-heptadecenyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyl group, acis-9-octadecenyl group (oleyl group), a cis-8,11-heptadecadienyl group,a cis-9,12-octadecadienyl group (linoleyl group), acis-10,13-nonadecadienyl group, or a cis-6,9,12-octadecatrienyl group(linolenyl group). The “C₁₀-C₂₄ alkenyl group” represented by L² ispreferably a decenyl group, an undecenyl group, a dodecenyl group, aheptadecenyl group, an octadecenyl group, a decadienyl group, anundecadienyl group, a dodecadienyl group, a heptadecadienyl group, anoctadecadienyl group, a nonadecadienyl group, a heptadecatrienyl group,an octadecatrienyl group, or a nonadecatrienyl group. L² is preferably acis-7-decenyl group, a (R)-11-acetyloxy-cis-8-heptadecenyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyl group, acis-9-octadecenyl group (oleyl group), a cis-8,11-heptadecadienyl group,a cis-9,12-octadecadienyl group (linoleyl group), acis-10,13-nonadecadienyl group, or a cis-6,9,12-octadecatrienyl group(linolenyl group).

The “C₃-C₂₄ alkynyl group” in the definitions of L¹ and L² refers to alinear alkynyl group having 3 to 24 carbon atoms. The “C₃-C₂₄ alkynylgroup” in the present application includes any of a C₃-C₂₄ alkadiynylgroup, a C₃-C₂₄ alkatriynyl group, and a C₃-C₂₄ alkatetraynyl group. TheC₃-C₂₄ alkynyl group is, for example, a propynyl group, a butynyl group,a pentynyl group, a hexynyl group, a heptynyl group, an octynyl group, anonynyl group, a decynyl group, an undecynyl group, a dodecynyl group, atridecynyl group, a tetradecynyl group, a pentadecynyl group, ahexadecynyl group, a heptadecynyl group, an octadecynyl group, anonadecynyl group, an icosynyl group, a henicosynyl group, a docosynylgroup, a tricosynyl group, or a tetracosynyl group and is preferably adecynyl group.

The “(C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) group” in the definitions ofL¹ and L² is, for example, a group represented by any of the followingstructural formulas:

The “(C₁₀-C₂₄ alkoxy)methyl group” in the definition of L² refers to agroup in which the “C₁₀-C₂₄ alkyl group” described above is bonded to anoxygen atom which is further bonded to a methyl group. Examples thereofcan include a decyloxymethyl group, an undecyloxymethyl group, adodecyloxymethyl group, a tridecyloxymethyl group, a tetradecyloxymethylgroup, a pentadecyloxymethyl group, a hexadecyloxymethyl group, aheptadecyloxymethyl group, an octadecyloxymethyl group, anonadecyloxymethyl group, an icosyloxymethyl group, a henicosyloxymethylgroup, a docosyloxymethyl group, a tricosyloxymethyl group, and atetracosyloxymethyl group. The “(C₁₀-C₂₄ alkoxy)methyl group” ispreferably a decyloxymethyl group, an undecyloxymethyl group, or adodecyloxymethyl group.

The “(C₁₀-C₂₄ alkenyl)oxymethyl group” in the definition of L² refers toa group in which the “C₁₀-C₂₄ alkenyl group” described above is bondedto an oxygen atom which is further bonded to a methyl group. The“(C₁₀-C₂₄ alkenyl)oxymethyl group” in the present application includesany of a (C₁₀-C₂₄ alkadienyl)oxymethyl group, a (C₁₀-C₂₄alkatrienyl)oxymethyl group, and a (C₁₀-C₂₄ alkatetraenyl)oxymethylgroup. Examples thereof can include a decenyloxymethyl group, anundecenyloxymethyl group, a dodecenyloxymethyl group, atridecenyloxymethyl group, a tetradecenyloxymethyl group, apentadecenyloxymethyl group, a hexadecenyloxymethyl group, aheptadecenyloxymethyl group, an octadecenyloxymethyl group, anonadecenyloxymethyl group, an icosenyloxymethyl group, ahenicosenyloxymethyl group, a docosenyloxymethyl group, atricosenyloxymethyl group, a tetracosenyloxymethyl group, adecadienyloxymethyl group, an undecadienyloxymethyl group, adodecadienyloxymethyl group, a tridecadienyloxymethyl group, atetradecadienyloxymethyl group, a pentadecadienyloxymethyl group, ahexadecadienyloxymethyl group, a heptadecadienyloxymethyl group, anoctadecadienyloxymethyl group, a nonadecadienyloxymethyl group, anicosadienyloxymethyl group, a henicosadienyloxymethyl group, adocosadienyloxymethyl group, a tricosadienyloxymethyl group, atetracosadienyloxymethyl group, a decatrienyloxymethyl group, anundecatrienyloxymethyl group, a dodecatrienyloxymethyl group, atridecatrienyloxymethyl group, a tetradecatrienyloxymethyl group, apentadecatrienyloxymethyl group, a hexadecatrienyloxymethyl group, aheptadecatrienyloxymethyl group, an octadecatrienyloxymethyl group, anonadecatrienyloxymethyl group, an icosatrienyloxymethyl group, ahenicosatrienyloxymethyl group, a docosatrienyloxymethyl group, atricosatrienyloxymethyl group, and a tetracosatrienyloxymethyl group.The “(C₁₀-C₂₄ alkenyl)oxymethyl group” is preferably a decenyloxymethylgroup, an undecenyloxymethyl group, a dodecenyloxymethyl group, aheptadecenyloxymethyl group, an octadecenyloxymethyl group, adecadienyloxymethyl group, an undecadienyloxymethyl group, adodecadienyloxymethyl group, a heptadecadienyloxymethyl group, anoctadecadienyloxymethyl group, a nonadecadienyloxymethyl group, aheptadecatrienyloxymethyl group, an octadecatrienyloxymethyl group, or anonadecatrienyloxymethyl group, more preferably a cis-7-decenyloxymethylgroup, a (R)-11-acetyloxy-cis-8-heptadecenyloxymethyl group, a(R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenyloxymethyl group,a cis-9-octadecenyloxymethyl group (oleyloxymethyl group), acis-8,11-heptadecadienyloxymethyl group, acis-9,12-octadecadienyloxymethyl group (linoleyloxymethyl group), acis-10,13-nonadecadienyloxymethyl group, or acis-6,9,12-octadecatrienyloxymethyl group (linolenyloxymethyl group).

The “(C₃-C₂₄ alkynyl)oxymethyl group” in the definition of L² refers toa group in which the “C₃-C₂₄ alkynyl group” described above is bonded toan oxygen atom which is further bonded to a methyl group. The “(C₃-C₂₄alkynyl)oxymethyl group” in the present application includes any of a(C₃-C₂₄ alkadiynyl)oxymethyl group, a (C₃-C₂₄ alkatriynyl)oxymethylgroup, and a (C₃-C₂₄ alkatetraynyl)oxymethyl group. The “(C₃-C₂₄alkynyl)oxymethyl group” is, for example, a propynyloxymethyl group, abutynyloxymethyl group, a pentynyloxymethyl group, a hexynyloxymethylgroup, a heptynyloxymethyl group, an octynyloxymethyl group, anonynyloxymethyl group, a decynyloxymethyl group, an undecynyloxymethylgroup, a dodecynyloxymethyl group, a tridecynyloxymethyl group, atetradecynyloxymethyl group, a pentadecynyloxymethyl group, ahexadecynyloxymethyl group, a heptadecynyloxymethyl group, anoctadecynyloxymethyl group, a nonadecynyloxymethyl group, anicosynyloxymethyl group, a henicosynyloxymethyl group, adocosynyloxymethyl group, a tricosynyloxymethyl group, or atetracosynyloxymethyl group and is preferably a decynyloxymethyl group.

The “(C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl)oxymethyl group” in thedefinition of L² refers to a group in which the “(C₁-C₁₀alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) group” described above is bonded to anoxygen atom which is further bonded to a methyl group.

When L¹ and L² each have one or more substituents selected fromsubstituent group β and the substituent(s) selected from substituentgroup β in L¹ and the substituent(s) selected from substituent group βin L² bind to each other to form a cyclic structure, substituent group βis preferably a C₂-C₆ alkanoyloxy group, more preferably a propionyloxygroup. More specifically, the substituent(s) selected from substituentgroup β in L¹ and the substituent(s) selected from substituent group βin L² bind to each other to form a group —OCOCH₂CH₂COO—.

The substituent group β in the present application is preferably thegroup consisting of a halogen atom, an oxo group, a cyano group, a C₁-C₆alkyl group, a C₁-C₆ halogenated alkyl group, a C₁-C₆ alkoxy group, aC₁-C₆ alkylsulfanyl group, a C₁-C₇ alkanoyl group, a C₁-C₇ alkanoyloxygroup, a C₃-C₇ alkoxyalkoxy group, a (C₁-C₆ alkoxy)carbonyl group, a(C₁-C₆ alkoxy)carboxyl group, a (C₁-C₆ alkoxy)carbamoyl group, and a(C₁-C₆ alkylamino)carboxyl group (substituent group β1), more preferablya C₂-C₅ alkanoyloxy group, further preferably an acetyloxy group or apropionyloxy group, particularly preferably an acetyloxy group.

The cationic lipid of the present invention can be converted to a“pharmacologically acceptable salt” by a standard method. Preferredexamples of such a salt can include: metal salts including alkali metalsalts such as a sodium salt, a potassium salt, and a lithium salt,alkaline earth metal salts such as a calcium salt and a magnesium salt,an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickelsalt, and a cobalt salt; amine salts including inorganic salts such asan ammonium salt and organic salts such as a t-octylamine salt, adibenzylamine salt, a morpholine salt, a glucosamine salt, aphenylglycine alkyl ester salt, an ethylenediamine salt, aN-methylglucamine salt, a guanidine salt, a diethylamine salt, atriethylamine salt, a dicyclohexylamine salt, aN,N′-dibenzylethylenediamine salt, a chloroprocaine salt, a procainesalt, a diethanolamine salt, a N-benzyl-phenethylamine salt, apiperazine salt, a tetramethylammonium salt, and atris(hydroxymethyl)aminomethane salt; inorganic acid salts such as ahydrohalide (e.g., a hydrofluoride, a hydrochloride, a hydrobromide, anda hydroiodide), a nitrate, a perchlorate, a sulfate, and a phosphate;organic acid salts such as lower alkanesulfonates (e.g., amethanesulfonate, a trifluoromethanesulfonate, and an ethanesulfonate),arylsulfonates (e.g., a benzenesulfonate and a p-toluenesulfonate), anacetate, a malate, a fumarate, a succinate, a citrate, a tartrate, anoxalate, and a maleate; and amino acid salts such as a glycine salt, alysine salt, an arginine salt, an ornithine salt, a glutamate, and anaspartate.

The cationic lipid of the present invention can also exist as a hydrateor a solvate. The present invention encompasses even such a hydrate orsolvate.

The cationic lipid of the present invention may have a stereoisomer, ageometric isomer, or an atropisomer. The present invention encompasseseven these isomers and mixtures of arbitrary isomers at an arbitraryratio, unless otherwise specified.

1-2. Specific Example of Cationic Lipid

Specific examples of the cationic lipid of the present invention caninclude compounds 1-1 to 1-481 described below in Table 1 and compounds2-1 to 2-570 described below in Table 2. In Tables 1 and 2, “C17-1”represents a cis-8-heptadecenyl group; “C18-1” represents acis-9-octadecenyl group (oleyl group); “C17-2” represents acis,cis-8,11-heptadecadienyl group; “Lin” represents acis,cis-9,12-octadecadienyl group (linoleyl group); “C19-2” represents acis,cis-10,13-nonadecadienyl group; “C17-31” represents acis,cis,cis-5,8,11-heptadecatrienyl group; “C17-32” represents acis,cis,cis-8,11,14-heptadecatrienyl group; “C17-33” represents a7-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]heptylgroup; “C17-A” represents a (R)-11-acetyloxy-cis-8-heptadecenyl group;“C17-H” represents a (R)-11-hexenyloxy-cis-8-heptadecenyl group;“C17-OH” represents a (R)-11-hydroxy-cis-8-heptadecenyl group; “C17-T”represents a (R)-11-(tetrahydro-2H-pyran-2-yloxy)-cis-8-heptadecenylgroup; “C17-T2” represents a(R)-11-(tetrahydro-2H-furan-2-yloxy)-cis-8-heptadecenyl group; “Me”represents a methyl group; “Et” represents an ethyl group; “Pr”represents a propyl group; “C10” represents a decyl group; “C11”represents an undecyl group; “C12” represents a dodecyl group; “C13”represents a tridecyl group; “C14” represents a tetradecyl group; “C15”represents a pentadecyl group; “C16” represents a hexadecyl group; “C17”represents a heptadecyl group; “C18” represents an octadecyl group;“C19” represents a nonadecyl group; “C20” represents an icosyl group;“C21” represents a henicosyl group; “C22” represents a docosyl group;“C23” represents a tricosyl group; “C24” represents a tetracosyl group;“C10-1” represents a cis-7-decenyl group; “C10-2” represents a 7-decynylgroup; “C17-O-Su-O-C17” represents a group in which(R)-11-hydroxy-cis-8-heptadecenyl groups are cross-linked via succinicacid; and “—” represents a single bond.

TABLE 1 [Formula 18]

Compound R¹ R² m Z L¹ L² R³ 1-1  Me Me 0 —(CH₂)₃— C16 C10 H 1-2  Me Me 0—(CH₂)₃— C16 C11 H 1-3  Me Me 0 —(CH₂)₃— C16 C12 H 1-4  Me Me 0 —(CH₂)₃—C16 C13 H 1-5  Me Me 0 —(CH₂)₃— C16 C14 H 1-6  Me Me 0 —(CH₂)₃— C16 C15H 1-7  Me Me 0 —(CH₂)₃— C16 C16 H 1-8  Me Me 0 —(CH₂)₃— C17 C10 H 1-9 Me Me 0 —(CH₂)₃— C17 C11 H 1-10  Me Me 0 —(CH₂)₃— C17 C12 H 1-11  Me Me0 —(CH₂)₃— C17 C13 H 1-12  Me Me 0 —(CH₂)₃— C17 C14 H 1-13  Me Me 0—(CH₂)₃— C17 C15 H 1-14  Me Me 0 —(CH₂)₃— C17 C16 H 1-15  Me Me 0—(CH₂)₃— C17 C17 H 1-16  Me Me 0 —(CH₂)₃— C17-1 C10 H 1-17  Me Et 0—(CH₂)₃— C17-1 C10 H 1-18  Me Pr 0 —(CH₂)₃— C17-1 C10 H 1-19  Et Et 0—(CH₂)₃— C17-1 C10 H 1-20  —(CH₂)₃— 0 —(CH₂)₃— C17-1 C10 H 1-21 —(CH₂)₄— 0 —(CH₂)₃— C17-1 C10 H 1-22  —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-1C10 H 1-23  Me Me 0 —CH₂CH(CH₃)CH₂— C17-1 C10 H 1-24  Me Me 0 —(CH₂)₄—C17-1 C10 H 1-25  Me Me 0 —(CH₂)₅— C17-1 C10 H 1-26  Me Me 1 —(CH₂)₃—C17-1 C10 H 1-27  Me Me 1 —(CH₂)₄— C17-1 C10 H 1-28  Me Me 0 —(CH₂)₃—C17-1 C10 Me 1-29  Me Et 0 —(CH₂)₃— C17-1 C10 Me 1-30  Me Me 0 —(CH₂)₃—C17-1 C10 Et 1-31  Me Me 0 —(CH₂)₃— C17-1 C10-1 H 1-32  Me Et 0 —(CH₂)₃—C17-1 C10-1 H 1-33  Me Pr 0 —(CH₂)₃— C17-1 C10-1 H 1-34  Et Et 0—(CH₂)₃— C17-1 C10-1 H 1-35  —(CH₂)₃— 0 —(CH₂)₃— C17-1 C10-1 H 1-36 —(CH₂)₄— 0 —(CH₂)₃— C17-1 C10-1 H 1-37  —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-1C10-1 H 1-38  Me Me 0 —CH₂CH(CH₃)CH₂— C17-1 C10-1 H 1-39  Me Me 0—(CH₂)₄— C17-1 C10-1 H 1-40  Me Me 0 —(CH₂)₅— C17-1 C10-1 H 1-41  Me Me1 —(CH₂)₃— C17-1 C10-1 H 1-42  Me Me 1 —(CH₂)₄— C17-1 C10-1 H 1-43  MeMe 0 —(CH₂)₃— C17-1 C11 H 1-44  Me Me 0 —(CH₂)₃— C17-1 C12 H 1-45  Me Me0 —(CH₂)₃— C17-1 C13 H 1-46  Me Me 0 —(CH₂)₃— C17-1 C14 H 1-47  Me Me 0—(CH₂)₃— C17-1 C15 H 1-48  Me Me 0 —(CH₂)₃— C17-1 C16 H 1-49  Me Me 0—(CH₂)₃— C17-1 C17 H 1-50  Me Me 0 —(CH₂)₃— C17-1 C17-1 H 1-51  Me Et 0—(CH₂)₃— C17-1 C17-1 H 1-52  Me Pr 0 —(CH₂)₃— C17-1 C17-1 H 1-53  Et Et0 —(CH₂)₃— C17-1 C17-1 H 1-54  —(CH₂)₃— 0 —(CH₂)₃— C17-1 C17-1 H 1-55 —(CH₂)₄— 0 —(CH₂)₃— C17-1 C17-1 H 1-56  —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-1C17-1 H 1-57  Me Me 0 —CH₂CH(CH₃)CH₂— C17-1 C17-1 H 1-58  Me Me 0—(CH₂)₄— C17-1 C17-1 H 1-59  Me Me 0 —(CH₂)₅— C17-1 C17-1 H 1-60  Me Me1 —(CH₂)₃— C17-1 C17-1 H 1-61  Me Me 1 —(CH₂)₄— C17-1 C17-1 H 1-62  MeMe 0 —(CH₂)₃— C17-1 C17-1 Me 1-63  Me Et 0 —(CH₂)₃— C17-1 C17-1 Me 1-64 Me Me 0 —(CH₂)₃— C17-1 C17-1 Et 1-65  Me Me 0 —(CH₂)₃— C17-1 C18 H 1-66 Me Me 0 —(CH₂)₃— C17-1 C19 H 1-67  Me Me 0 —(CH₂)₃— C17-1 C20 H 1-68  MeMe 0 —(CH₂)₃— C17-1 C21 H 1-69  Me Me 0 —(CH₂)₃— C17-1 C22 H 1-70  Me Me0 —(CH₂)₃— C17-1 C23 H 1-71  Me Me 0 —(CH₂)₃— C17-1 C24 H 1-72  Me Me 0—(CH₂)₃— C17-2 C10 H 1-73  Me Et 0 —(CH₂)₃— C17-2 C10 H 1-74  Me Pr 0—(CH₂)₃— C17-2 C10 H 1-75  Et Et 0 —(CH₂)₃— C17-2 C10 H 1-76  —(CH₂)₃— 0—(CH₂)₃— C17-2 C10 H 1-77  —(CH₂)₄— 0 —(CH₂)₃— C17-2 C10 H 1-78 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-2 C10 H 1-79  Me Me 0 —CH₂CH(CH₃)CH₂—C17-2 C10 H 1-80  Me Me 0 —(CH₂)₄— C17-2 C10 H 1-81  Me Me 0 —(CH₂)₅—C17-2 C10 H 1-82  Me Me 1 —(CH₂)₃— C17-2 C10 H 1-83  Me Me 1 —(CH₂)₄—C17-2 C10 H 1-84  Me Me 0 —(CH₂)₃— C17-2 C10 Me 1-85  Me Et 0 —(CH₂)₃—C17-2 C10 Me 1-86  Me Me 0 —(CH₂)₃— C17-2 C10 Et 1-87  Me Me 0 —(CH₂)₃—C17-2 C10-1 H 1-88  Me Et 0 —(CH₂)₃— C17-2 C10-1 H 1-89  Me Pr 0—(CH₂)₃— C17-2 C10-1 H 1-90  Et Et 0 —(CH₂)₃— C17-2 C10-1 H 1-91 —(CH₂)₃— 0 —(CH₂)₃— C17-2 C10-1 H 1-92  —(CH₂)₄— 0 —(CH₂)₃— C17-2 C10-1H 1-93  —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-2 C10-1 H 1-94  Me Me 0—CH₂CH(CH₃)CH₂— C17-2 C10-1 H 1-95  Me Me 0 —(CH₂)₄— C17-2 C10-1 H 1-96 Me Me 0 —(CH₂)₅— C17-2 C10-1 H 1-97  Me Me 1 —(CH₂)₃— C17-2 C10-1 H1-98  Me Me 1 —(CH₂)₄— C17-2 C10-1 H 1-99  Me Me 0 —(CH₂)₃— C17-2 C10-2H 1-100 Me Et 0 —(CH₂)₃— C17-2 C10-2 H 1-101 Me Pr 0 —(CH₂)₃— C17-2C10-2 H 1-102 Et Et 0 —(CH₂)₃— C17-2 C10-2 H 1-103 —(CH₂)₃— 0 —(CH₂)₃—C17-2 C10-2 H 1-104 —(CH₂)₄— 0 —(CH₂)₃— C17-2 C10-2 H 1-105—(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-2 C10-2 H 1-106 Me Me 0 —CH₂CH(CH₃)CH₂—C17-2 C10-2 H 1-107 Me Me 0 —(CH₂)₄— C17-2 C10-2 H 1-108 Me Me 0—(CH₂)₅— C17-2 C10-2 H 1-109 Me Me 1 —(CH₂)₃— C17-2 C10-2 H 1-110 Me Me1 —(CH₂)₄— C17-2 C10-2 H 1-111 Me Me 0 —(CH₂)₃— C17-2 C11 H 1-112 Me Me0 —(CH₂)₃— C17-2 C11 H 1-113 Me Me 0 —(CH₂)₃— C17-2 C13 H 1-114 Me Me 0—(CH₂)₃— C17-2 C14 H 1-115 Me Me 0 —(CH₂)₃— C17-2 C15 H 1-116 Me Me 0—(CH₂)₃— C17-2 C16 H 1-117 Me Me 0 —(CH₂)₃— C17-2 C17 H 1-118 Me Me 0—(CH₂)₃— C17-2 C17-2 H 1-119 Me Me 0 —(CH₂)₃— C17-2 C17-2 H 1-120 Me Me0 —(CH₂)₃— C17-2 C17-2 H 1-121 Me Et 0 —(CH₂)₃— C17-2 C17-2 H 1-122 MeEt 0 —(CH₂)₃— C17-2 C17-2 H 1-123 Me Pr 0 —(CH₂)₃— C17-2 C17-2 H 1-124Et Et 0 —(CH₂)₃— C17-2 C17-2 H 1-125 —(CH₂)₃— 0 —(CH₂)₃— C17-2 C17-2 H1-126 —(CH₂)₄— 0 —(CH₂)₃— C17-2 C17-2 H 1-127 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃—C17-2 C17-2 H 1-128 Me Me 0 —CH₂CH(CH₃)CH₂— C17-2 C17-2 H 1-129 Me Me 0—(CH₂)₄— C17-2 C17-2 H 1-130 Me Me 0 —(CH₂)₅— C17-2 C17-2 H 1-131 Me Me1 —(CH₂)₃— C17-2 C17-2 H 1-132 Me Me 1 —(CH₂)₄— C17-2 C17-2 H 1-133 MeMe 0 —(CH₂)₃— C17-2 C18 H 1-134 Me Me 0 —(CH₂)₃— C17-2 C19 H 1-135 Me Me0 —(CH₂)₃— C17-2 C20 H 1-136 Me Me 0 —(CH₂)₃— C17-2 C21 H 1-137 Me Me 0—(CH₂)₃— C17-2 C22 H 1-138 Me Me 0 —(CH₂)₃— C17-2 C23 H 1-139 Me Me 0—(CH₂)₃— C17-2 C24 H 1-140 Me Me 0 —(CH₂)₃— C17-31 C10 H 1-141 Me Et 0—(CH₂)₃— C17-31 C10 H 1-142 Me Pr 0 —(CH₂)₃— C17-31 C10 H 1-143 Et Et 0—(CH₂)₃— C17-31 C10 H 1-144 —(CH₂)₃— 0 —(CH₂)₃— C17-31 C10 H 1-145—(CH₂)₄— 0 —(CH₂)₃— C17-31 C10 H 1-146 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-31C10 H 1-147 Me Me 0 —CH₂CH(CH₃)CH₂— C17-31 C10 H 1-148 Me Me 0 —(CH₂)₄—C17-31 C10 H 1-149 Me Me 0 —(CH₂)₅— C17-31 C10 H 1-150 Me Me 1 —(CH₂)₃—C17-31 C10 H 1-151 Me Me 1 —(CH₂)₄— C17-31 C10 H 1-152 Me Me 0 —(CH₂)₃—C17-31 C17-31 H 1-153 Me Et 0 —(CH₂)₃— C17-31 C17-31 H 1-154 Me Pr 0—(CH₂)₃— C17-31 C17-31 H 1-155 Et Et 0 —(CH₂)₃— C17-31 C17-31 H 1-156—(CH₂)₃— 0 —(CH₂)₃— C17-31 C17-31 H 1-157 —(CH₂)₄— 0 —(CH₂)₃— C17-31C17-31 H 1-158 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-31 C17-31 H 1-159 Me Me 0—CH₂CH(CH₃)CH₂— C17-31 C17-31 H 1-160 Me Me 0 —(CH₂)₄— C17-31 C17-31 H1-161 Me Me 0 —(CH₂)₅— C17-31 C17-31 H 1-162 Me Me 1 —(CH₂)₃— C17-31C17-31 H 1-163 Me Me 1 —(CH₂)₄— C17-31 C17-31 H 1-164 Me Me 0 —(CH₂)₃—C17-32 C10 H 1-165 Me Et 0 —(CH₂)₃— C17-32 C10 H 1-166 Me Pr 0 —(CH₂)₃—C17-32 C10 H 1-167 Et Et 0 —(CH₂)₃— C17-32 C10 H 1-168 —(CH₂)₃— 0—(CH₂)₃— C17-32 C10 H 1-169 —(CH₂)₄— 0 —(CH₂)₃— C17-32 C10 H 1-170—(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-32 C10 H 1-171 Me Me 0 —CH₂CH(CH₃)CH₂—C17-32 C10 H 1-172 Me Me 0 —(CH₂)₄— C17-32 C10 H 1-173 Me Me 0 —(CH₂)₅—C17-32 C10 H 1-174 Me Me 1 —(CH₂)₃— C17-32 C10 H 1-175 Me Me 1 —(CH₂)₄—C17-32 C10 H 1-176 Me Me 0 —(CH₂)₃— C17-32 C17-32 H 1-177 Me Et 0—(CH₂)₃— C17-32 C17-32 H 1-178 Me Pr 0 —(CH₂)₃— C17-32 C17-32 H 1-179 EtEt 0 —(CH₂)₃— C17-32 C17-32 H 1-180 —(CH₂)₃— 0 —(CH₂)₃— C17-32 C17-32 H1-181 —(CH₂)₄— 0 —(CH₂)₃— C17-32 C17-32 H 1-182 —(CH₂)₂O(CH₂)₂— 0—(CH₂)₃— C17-32 C17-32 H 1-183 Me Me 0 —CH₂CH(CH₃)CH₂— C17-32 C17-32 H1-184 Me Me 0 —(CH₂)₄— C17-32 C17-32 H 1-185 Me Me 0 —(CH₂)₅— C17-32C17-32 H 1-186 Me Me 1 —(CH₂)₃— C17-32 C17-32 H 1-187 Me Me 1 —(CH₂)₄—C17-32 C17-32 H 1-188 Me Me 0 —(CH₂)₃— C17-33 C10 H 1-189 Me Et 0—(CH₂)₃— C17-33 C10 H 1-190 Me Pr 0 —(CH₂)₃— C17-33 C10 H 1-191 Et Et 0—(CH₂)₃— C17-33 C10 H 1-192 —(CH₂)₃— 0 —(CH₂)₃— C17-33 C10 H 1-193—(CH₂)₄— 0 —(CH₂)₃— C17-33 C10 H 1-194 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-33C10 H 1-195 Me Me 0 —CH₂CH(CH₃)CH₂— C17-33 C10 H 1-196 Me Me 0 —(CH₂)₄—C17-33 C10 H 1-197 Me Me 0 —(CH₂)₅— C17-33 C10 H 1-198 Me Me 1 —(CH₂)₃—C17-33 C10 H 1-199 Me Me 1 —(CH₂)₄— C17-33 C10 H 1-200 Me Me 0 —(CH₂)₃—C17-33 C17-33 H 1-201 Me Et 0 —(CH₂)₃— C17-33 C17-33 H 1-202 Me Pr 0—(CH₂)₃— C17-33 C17-33 H 1-203 Et Et 0 —(CH₂)₃— C17-33 C17-33 H 1-204—(CH₂)₃— 0 —(CH₂)₃— C17-33 C17-33 H 1-205 —(CH₂)₄— 0 —(CH₂)₃— C17-33C17-33 H 1-206 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-33 C17-33 H 1-207 Me Me 0—CH₂CH(CH₃)CH₂— C17-33 C17-33 H 1-208 Me Me 0 —(CH₂)₄— C17-33 C17-33 H1-209 Me Me 0 —(CH₂)₅— C17-33 C17-33 H 1-210 Me Me 1 —(CH₂)₃— C17-33C17-33 H 1-211 Me Me 1 —(CH₂)₄— C17-33 C17-33 H 1-212 Me Me 0 —(CH₂)₃—C17-A C10 H 1-213 Me Et 0 —(CH₂)₃— C17-A C10 H 1-214 Me Pr 0 —(CH₂)₃—C17-A C10 H 1-215 Et Et 0 —(CH₂)₃— C17-A C10 H 1-216 —(CH₂)₃— 0 —(CH₂)₃—C17-A C10 H 1-217 —(CH₂)₄— 0 —(CH₂)₃— C17-A C10 H 1-218 —(CH₂)₂O(CH₂)₂—0 —(CH₂)₃— C17-A C10 H 1-219 Me Me 0 —CH₂CH(CH₃)CH₂— C17-A C10 H 1-220Me Me 0 —(CH₂)₄— C17-A C10 H 1-221 Me Me 0 —(CH₂)₅— C17-A C10 H 1-222 MeMe 1 —(CH₂)₃— C17-A C10 H 1-223 Me Me 1 —(CH₂)₄— C17-A C10 H 1-224 Me Me0 —(CH₂)₃— C17-A C11 H 1-225 Me Me 0 —(CH₂)₃— C17-A C12 H 1-226 Me Me 0—(CH₂)₃— C17-A C13 H 1-227 Me Me 0 —(CH₂)₃— C17-A C14 H 1-228 Me Me 0—(CH₂)₃— C17-A C15 H 1-229 Me Me 0 —(CH₂)₃— C17-A C16 H 1-230 Me Me 0—(CH₂)₃— C17-A C17 H 1-231 Me Me 0 —(CH₂)₃— C17-A C17-1 H 1-232 Me Me 0—(CH₂)₃— C17-A C17-2 H 1-233 Me Me 0 —(CH₂)₃— C17-A C17-A H 1-234 Me Et0 —(CH₂)₃— C17-A C17-A H 1-235 Me Pr 0 —(CH₂)₃— C17-A C17-A H 1-236 EtEt 0 —(CH₂)₃— C17-A C17-A H 1-237 —(CH₂)₃— 0 —(CH₂)₃— C17-A C17-A H1-238 —(CH₂)₄— 0 —(CH₂)₃— C17-A C17-A H 1-239 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃—C17-A C17-A H 1-240 Me Me 0 —CH₂CH(CH₃)CH₂— C17-A C17-A H 1-241 Me Me 0—(CH₂)₄— C17-A C17-A H 1-242 Me Me 0 —(CH₂)₅— C17-A C17-A H 1-243 Me Me1 —(CH₂)₃— C17-A C17-A H 1-244 Me Me 1 —(CH₂)₄— C17-A C17-A H 1-245 MeMe 0 —(CH₂)₃— C17-A C18 H 1-246 Me Me 0 —(CH₂)₃— C17-A C18-1 H 1-247 MeMe 0 —(CH₂)₃— C17-A C19 H 1-248 Me Me 0 —(CH₂)₃— C17-A C19-2 H 1-249 MeMe 0 —(CH₂)₃— C17-A C20 H 1-250 Me Me 0 —(CH₂)₃— C17-A C21 H 1-251 Me Me0 —(CH₂)₃— C17-A C22 H 1-252 Me Me 0 —(CH₂)₃— C17-A C23 H 1-253 Me Me 0—(CH₂)₃— C17-A C24 H 1-254 Me Me 0 —(CH₂)₃— C17-A Lin H 1-255 Me Me 0—(CH₂)₃— C17-H C10 H 1-256 Me Et 0 —(CH₂)₃— C17-H C10 H 1-257 Me Pr 0—(CH₂)₃— C17-H C10 H 1-258 Et Et 0 —(CH₂)₃— C17-H C10 H 1-259 —(CH₂)₃— 0—(CH₂)₃— C17-H C10 H 1-260 —(CH₂)₄— 0 —(CH₂)₃— C17-H C10 H 1-261—(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-H C10 H 1-262 Me Me 0 —CH₂CH(CH₃)CH₂—C17-H C10 H 1-263 Me Me 0 —(CH₂)₄— C17-H C10 H 1-264 Me Me 0 —(CH₂)₅—C17-H C10 H 1-265 Me Me 1 —(CH₂)₃— C17-H C10 H 1-266 Me Me 1 —(CH₂)₄—C17-H C10 H 1-267 Me Me 0 —(CH₂)₃— C17-H C11 H 1-268 Me Me 0 —(CH₂)₃—C17-H C12 H 1-269 Me Me 0 —(CH₂)₃— C17-H C13 H 1-270 Me Me 0 —(CH₂)₃—C17-H C14 H 1-271 Me Me 0 —(CH₂)₃— C17-H C15 H 1-272 Me Me 0 —(CH₂)₃—C17-H C16 H 1-273 Me Me 0 —(CH₂)₃— C17-H C17 H 1-274 Me Me 0 —(CH₂)₃—C17-H C17-1 H 1-275 Me Me 0 —(CH₂)₃— C17-H C17-2 H 1-276 Me Me 0—(CH₂)₃— C17-H C17-H H 1-277 Me Et 0 —(CH₂)₃— C17-H C17-H H 1-278 Me Pr0 —(CH₂)₃— C17-H C17-H H 1-279 Et Et 0 —(CH₂)₃— C17-H C17-H H 1-280—(CH₂)₃— 0 —(CH₂)₃— C17-H C17-H H 1-281 —(CH₂)₄— 0 —(CH₂)₃— C17-H C17-HH 1-282 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-H C17-H H 1-283 Me Me 0—CH₂CH(CH₃)CH₂— C17-H C17-H H 1-284 Me Me 0 —(CH₂)₄— C17-H C17-H H 1-285Me Me 0 —(CH₂)₅— C17-H C17-H H 1-286 Me Me 1 —(CH₂)₃— C17-H C17-H H1-287 Me Me 1 —(CH₂)₄— C17-H C17-H H 1-288 Me Me 0 —(CH₂)₃— C17-H C18 H1-289 Me Me 0 —(CH₂)₃— C17-H C18-1 H 1-290 Me Me 0 —(CH₂)₃— C17-H C19 H1-291 Me Me 0 —(CH₂)₃— C17-H C19-2 H 1-292 Me Me 0 —(CH₂)₃— C17-H C20 H1-293 Me Me 0 —(CH₂)₃— C17-H C21 H 1-294 Me Me 0 —(CH₂)₃— C17-H C22 H1-295 Me Me 0 —(CH₂)₃— C17-H C23 H 1-296 Me Me 0 —(CH₂)₃— C17-H C24 H1-297 Me Me 0 —(CH₂)₃— C17-H Lin H 1-298 Me Me 0 —(CH₂)₃— C17-OH C10 H1-299 Me Me 0 —(CH₂)₃— C17-OH C11 H 1-300 Me Me 0 —(CH₂)₃— C17-OH C12 H1-301 Me Me 0 —(CH₂)₃— C17-OH C13 H 1-302 Me Me 0 —(CH₂)₃— C17-OH C14 H1-303 Me Me 0 —(CH₂)₃— C17-OH C15 H 1-304 Me Me 0 —(CH₂)₃— C17-OH C16 H1-305 Me Me 0 —(CH₂)₃— C17-OH C17 H 1-306 Me Me 0 —(CH₂)₃— C17-OH C17-1H 1-307 Me Me 0 —(CH₂)₃— C17-OH C17-2 H 1-308 Me Me 0 —(CH₂)₃— C17-OHC17-OH H 1-309 Me Me 0 —(CH₂)₃— C17-OH C18 H 1-310 Me Me 0 —(CH₂)₃—C17-OH C18-1 H 1-311 Me Me 0 —(CH₂)₃— C17-OH C19 H 1-312 Me Me 0—(CH₂)₃— C17-OH C19-2 H 1-313 Me Me 0 —(CH₂)₃— C17-OH C20 H 1-314 Me Me0 —(CH₂)₃— C17-OH C21 H 1-315 Me Me 0 —(CH₂)₃— C17-OH C22 H 1-316 Me Me0 —(CH₂)₃— C17-OH C23 H 1-317 Me Me 0 —(CH₂)₃— C17-OH C24 H 1-318 Me Me0 —(CH₂)₃— C17-OH Lin H 1-319 Me Me 0 —(CH₂)₃— C17-O-Su-O-C17 H 1-320 MeMe 0 —(CH₂)₃— C17-O-Su-O-C17 H 1-321 Me Me 0 —(CH₂)₃— C17-O-Su-O-C17 H1-322 Me Et 0 —(CH₂)₃— C17-O-Su-O-C17 H 1-323 Me Et 0 —(CH₂)₃—C17-O-Su-O-C17 H 1-324 Me Pr 0 —(CH₂)₃— C17-O-Su-O-C17 H 1-325 Et Et 0—(CH₂)₃— C17-O-Su-O-C17 H 1-326 —(CH₂)₃— 0 —(CH₂)₃— C17-O-Su-O-C17 H1-327 —(CH₂)₄— 0 —(CH₂)₃— C17-O-Su-O-C17 H 1-328 —(CH₂)₂O(CH₂)₂— 0—(CH₂)₃— C17-O-Su-O-C17 H 1-329 Me Me 0 —CH₂CH(CH₃)CH₂— C17-O-Su-O-C17 H1-330 Me Me 0 —(CH₂)₄— C17-O-Su-O-C17 H 1-331 Me Me 0 —(CH₂)₅—C17-O-Su-O-C17 H 1-332 Me Me 1 —(CH₂)₃— C17-O-Su-O-C17 H 1-333 Me Me 1—(CH₂)₄— C17-O-Su-O-C17 H 1-334 Me Me 0 —(CH₂)₃— C17-T C10 H 1-335 Me Et0 —(CH₂)₃— C17-T C10 H 1-336 Me Pr 0 —(CH₂)₃— C17-T C10 H 1-337 Et Et 0—(CH₂)₃— C17-T C10 H 1-338 —(CH₂)₃— 0 —(CH₂)₃— C17-T C10 H 1-339—(CH₂)₄— 0 —(CH₂)₃— C17-T C10 H 1-340 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-TC10 H 1-341 Me Me 0 —CH₂CH(CH₃)CH₂— C17-T C10 H 1-342 Me Me 0 —(CH₂)₄—C17-T C10 H 1-343 Me Me 0 —(CH₂)₅— C17-T C10 H 1-344 Me Me 1 —(CH₂)₃—C17-T C10 H 1-345 Me Me 1 —(CH₂)₄— C17-T C10 H 1-346 Me Me 0 —(CH₂)₃—C17-T C11 H 1-347 Me Me 0 —(CH₂)₃— C17-T C12 H 1-348 Me Me 0 —(CH₂)₃—C17-T C13 H 1-349 Me Me 0 —(CH₂)₃— C17-T C14 H 1-350 Me Me 0 —(CH₂)₃—C17-T C15 H 1-351 Me Me 0 —(CH₂)₃— C17-T C16 H 1-352 Me Me 0 —(CH₂)₃—C17-T C17 H 1-353 Me Me 0 —(CH₂)₃— C17-T C17-1 H 1-354 Me Me 0 —(CH₂)₃—C17-T C17-2 H 1-355 Me Me 0 —(CH₂)₃— C17-T C17-T H 1-356 Me Et 0—(CH₂)₃— C17-T C17-T H 1-357 Me Pr 0 —(CH₂)₃— C17-T C17-T H 1-358 Et Et0 —(CH₂)₃— C17-T C17-T H 1-359 —(CH₂)₃— 0 —(CH₂)₃— C17-T C17-T H 1-360—(CH₂)₄— 0 —(CH₂)₃— C17-T C17-T H 1-361 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-TC17-T H 1-362 Me Me 0 —CH₂CH(CH₃)CH₂— C17-T C17-T H 1-363 Me Me 0—(CH₂)₄— C17-T C17-T H 1-364 Me Me 0 —(CH₂)₅— C17-T C17-T H 1-365 Me Me1 —(CH₂)₃— C17-T C17-T H 1-366 Me Me 1 —(CH₂)₄— C17-T C17-T H 1-367 MeMe 0 —(CH₂)₃— C17-T C18 H 1-368 Me Me 0 —(CH₂)₃— C17-T C18-1 H 1-369 MeMe 0 —(CH₂)₃— C17-T C19 H 1-370 Me Me 0 —(CH₂)₃— C17-T C19-2 H 1-371 MeMe 0 —(CH₂)₃— C17-T C20 H 1-372 Me Me 0 —(CH₂)₃— C17-T C21 H 1-373 Me Me0 —(CH₂)₃— C17-T C22 H 1-374 Me Me 0 —(CH₂)₃— C17-T C23 H 1-375 Me Me 0—(CH₂)₃— C17-T C24 H 1-376 Me Me 0 —(CH₂)₃— C17-T Lin H 1-377 Me Me 0—(CH₂)₃— C17-T2 C10 H 1-378 Me Et 0 —(CH₂)₃— C17-T2 C10 H 1-379 Me Pr 0—(CH₂)₃— C17-T2 C10 H 1-380 Et Et 0 —(CH₂)₃— C17-T2 C10 H 1-381 —(CH₂)₃—0 —(CH₂)₃— C17-T2 C10 H 1-382 —(CH₂)₄— 0 —(CH₂)₃— C17-T2 C10 H 1-383—(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-T2 C10 H 1-384 Me Me 0 —CH₂CH(CH₃)CH₂—C17-T2 C10 H 1-385 Me Me 0 —(CH₂)₄— C17-T2 C10 H 1-386 Me Me 0 —(CH₂)₅—C17-T2 C10 H 1-387 Me Me 1 —(CH₂)₃— C17-T2 C10 H 1-388 Me Me 1 —(CH₂)₄—C17-T2 C10 H 1-389 Me Me 0 —(CH₂)₃— C17-T2 C11 H 1-390 Me Me 0 —(CH₂)₃—C17-T2 C12 H 1-391 Me Me 0 —(CH₂)₃— C17-T2 C13 H 1-392 Me Me 0 —(CH₂)₃—C17-T2 C14 H 1-393 Me Me 0 —(CH₂)₃— C17-T2 C15 H 1-394 Me Me 0 —(CH₂)₃—C17-T2 C16 H 1-395 Me Me 0 —(CH₂)₃— C17-T2 C17 H 1-396 Me Me 0 —(CH₂)₃—C17-T2 C17-1 H 1-397 Me Me 0 —(CH₂)₃— C17-T2 C17-2 H 1-398 Me Me 0—(CH₂)₃— C17-T2 C17-T2 H 1-399 Me Et 0 —(CH₂)₃— C17-T2 C17-T2 H 1-400 MePr 0 —(CH₂)₃— C17-T2 C17-T2 H 1-401 Et Et 0 —(CH₂)₃— C17-T2 C17-T2 H1-402 —(CH₂)₃— 0 —(CH₂)₃— C17-T2 C17-T2 H 1-403 —(CH₂)₄— 0 —(CH₂)₃—C17-T2 C17-T2 H 1-404 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C17-T2 C17-T2 H 1-405Me Me 0 —CH₂CH(CH₃)CH₂— C17-T2 C17-T2 H 1-406 Me Me 0 —(CH₂)₄— C17-T2C17-T2 H 1-407 Me Me 0 —(CH₂)₅— C17-T2 C17-T2 H 1-408 Me Me 1 —(CH₂)₃—C17-T2 C17-T2 H 1-409 Me Me 1 —(CH₂)₄— C17-T2 C17-T2 H 1-410 Me Me 0—(CH₂)₃— C17-T2 C18 H 1-411 Me Me 0 —(CH₂)₃— C17-T2 C18-1 H 1-412 Me Me0 —(CH₂)₃— C17-T2 C19 H 1-413 Me Me 0 —(CH₂)₃— C17-T2 C19-2 H 1-414 MeMe 0 —(CH₂)₃— C17-T2 C20 H 1-415 Me Me 0 —(CH₂)₃— C17-T2 C21 H 1-416 MeMe 0 —(CH₂)₃— C17-T2 C22 H 1-417 Me Me 0 —(CH₂)₃— C17-T2 C23 H 1-418 MeMe 0 —(CH₂)₃— C17-T2 C24 H 1-419 Me Me 0 —(CH₂)₃— C17-T2 Lin H 1-420 MeMe 0 —(CH₂)₃— C18 C10 H 1-421 Me Me 0 —(CH₂)₃— C18 C11 H 1-422 Me Me 0—(CH₂)₃— C18 C12 H 1-423 Me Me 0 —(CH₂)₃— C18 C13 H 1-424 Me Me 0—(CH₂)₃— C18 C14 H 1-425 Me Me 0 —(CH₂)₃— C18 C15 H 1-426 Me Me 0—(CH₂)₃— C18 C16 H 1-427 Me Me 0 —(CH₂)₃— C18 C17 H 1-428 Me Me 0—(CH₂)₃— C18 C18 H 1-429 Me Me 0 —(CH₂)₃— C18-1 C18-1 H 1-430 Me Et 0—(CH₂)₃— C18-1 C18-1 H 1-431 Me Pr 0 —(CH₂)₃— C18-1 C18-1 H 1-432 Et Et0 —(CH₂)₃— C18-1 C18-1 H 1-433 —(CH₂)₃— 0 —(CH₂)₃— C18-1 C18-1 H 1-434—(CH₂)₄— 0 —(CH₂)₃— C18-1 C18-1 H 1-435 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C18-1C18-1 H 1-436 Me Me 0 —CH₂CH(CH₃)CH₂— C18-1 C18-1 H 1-437 Me Me 0—(CH₂)₄— C18-1 C18-1 H 1-438 Me Me 0 —(CH₂)₅— C18-1 C18-1 H 1-439 Me Me1 —(CH₂)₃— C18-1 C18-1 H 1-440 Me Me 1 —(CH₂)₄— C18-1 C18-1 H 1-441 MeMe 0 —(CH₂)₃— C18-1 C18-1 Me 1-442 Me Et 0 —(CH₂)₃— C18-1 C18-1 Me 1-443Me Me 0 —(CH₂)₃— C18-1 C18-1 Et 1-444 Me Me 0 —(CH₂)₃— C19-2 C10 H 1-445Me Et 0 —(CH₂)₃— C19-2 C10 H 1-446 Me Pr 0 —(CH₂)₃— C19-2 C10 H 1-447 EtEt 0 —(CH₂)₃— C19-2 C10 H 1-448 —(CH₂)₃— 0 —(CH₂)₃— C19-2 C10 H 1-449—(CH₂)₄— 0 —(CH₂)₃— C19-2 C10 H 1-450 —(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— C19-2C10 H 1-451 Me Me 0 —CH₂CH(CH₃)CH₂— C19-2 C10 H 1-452 Me Me 0 —(CH₂)₄—C19-2 C10 H 1-453 Me Me 0 —(CH₂)₅— C19-2 C10 H 1-454 Me Me 1 —(CH₂)₃—C19-2 C10 H 1-455 Me Me 1 —(CH₂)₄— C19-2 C10 H 1-456 Me Me 0 —(CH₂)₃—C19-2 C10 Me 1-457 Me Et 0 —(CH₂)₃— C19-2 C10 Me 1-458 Me Me 0 —(CH₂)₃—C19-2 C10 Et 1-459 Me Me 1 —(CH₂)₃— Lin C10 H 1-460 Me Et 1 —(CH₂)₃— LinC10 H 1-461 Me Pr 1 —(CH₂)₃— Lin C10 H 1-462 Et Et 1 —(CH₂)₃— Lin C10 H1-463 —(CH₂)₃— 1 —(CH₂)₃— Lin C10 H 1-464 —(CH₂)₄— 1 —(CH₂)₃— Lin C10 H1-465 —(CH₂)₂O(CH₂)₂— 1 —(CH₂)₃— Lin C10 H 1-466 Me Me 1 —(CH₂)₄— LinC10 H 1-467 Me Me 0 —(CH₂)₃— Lin Lin H 1-468 Me Et 0 —(CH₂)₃— Lin Lin H1-469 Me Pr 0 —(CH₂)₃— Lin Lin H 1-470 Et Et 0 —(CH₂)₃— Lin Lin H 1-471—(CH₂)₃— 0 —(CH₂)₃— Lin Lin H 1-472 —(CH₂)₄— 0 —(CH₂)₃— Lin Lin H 1-473—(CH₂)₂O(CH₂)₂— 0 —(CH₂)₃— Lin Lin H 1-474 Me Me 0 —CH₂CH(CH₃)CH₂— LinLin H 1-475 Me Me 0 —(CH₂)₄— Lin Lin H 1-476 Me Me 0 —(CH₂)₅— Lin Lin H1-477 Me Me 1 —(CH₂)₃— Lin Lin H 1-478 Me Me 1 —(CH₂)₄— Lin Lin H 1-479Me Me 0 —(CH₂)₃— Lin Lin Me 1-480 Me Et 0 —(CH₂)₃— Lin Lin Me 1-481 MeMe 0 —(CH₂)₃— Lin Lin Et

TABLE 2 [Formula 19]

Compound R² p q m Z L¹ L² R³ 2-1 Me 0 2 0 — C17-1 C10 H 2-2 Me 0 3 0 —C17-1 C10 H 2-3 Me 0 4 0 — C17-1 C10 H 2-4 Me 1 2 0 — C17-1 C10 H 2-5 Me1 3 0 — C17-1 C10 H 2-6 Me 2 2 0 — C17-1 C10 H 2-7 Me 0 2 0 —CH₂— C17-1C10 H 2-8 Me 0 3 0 —CH₂— C17-1 C10 H 2-9 Me 0 4 0 —CH₂— C17-1 C10 H 2-10Me 1 1 0 —CH₂— C17-1 C10 H 2-11 Me 1 2 0 —CH₂— C17-1 C10 H 2-12 Me 1 3 0—CH₂— C17-1 C10 H 2-13 Me 2 2 0 —CH₂— C17-1 C10 H 2-14 Me 0 2 0 —(CH₂)₂—C17-1 C10 H 2-15 Me 0 3 0 —(CH₂)₂— C17-1 C10 H 2-16 Me 0 4 0 —(CH₂)₂—C17-1 C10 H 2-17 Me 1 1 0 —(CH₂)₂— C17-1 C10 H 2-18 Me 1 2 0 —(CH₂)₂—C17-1 C10 H 2-19 Me 1 3 0 —(CH₂)₂— C17-1 C10 H 2-20 Me 2 2 0 —(CH₂)₂—C17-1 C10 H 2-21 Me 0 2 0 —(CH₂)₃— C17-1 C10 H 2-22 Me 0 3 0 —(CH₂)₃—C17-1 C10 H 2-23 Me 0 4 0 —(CH₂)₃— C17-1 C10 H 2-24 Me 1 1 0 —(CH₂)₃—C17-1 C10 H 2-25 Me 1 2 0 —(CH₂)₃— C17-1 C10 H 2-26 Me 1 3 0 —(CH₂)₃—C17-1 C10 H 2-27 Me 2 2 0 —(CH₂)₃— C17-1 C10 H 2-28 Me 1 3 0 — C17-1 C10Me 2-29 Me 2 2 0 — C17-1 C10 Me 2-30 Me 1 3 0 —CH₂— C17-1 C10 Me 2-31 Me0 2 0 — C17-1 C17-1 H 2-32 Me 0 3 0 — C17-1 C17-1 H 2-33 Me 0 4 0 —C17-1 C17-1 H 2-34 Me 1 2 0 — C17-1 C17-1 H 2-35 Me 1 3 0 — C17-1 C17-1H 2-36 Me 2 2 0 — C17-1 C17-1 H 2-37 Me 0 2 0 —CH₂— C17-1 C17-1 H 2-38Me 0 3 0 —CH₂— C17-1 C17-1 H 2-39 Me 0 4 0 —CH₂— C17-1 C17-1 H 2-40 Me 11 0 —CH₂— C17-1 C17-1 H 2-41 Me 1 2 0 —CH₂— C17-1 C17-1 H 2-42 Me 1 3 0—CH₂— C17-1 C17-1 H 2-43 Me 2 2 0 —CH₂— C17-1 C17-1 H 2-44 Me 0 2 0—(CH₂)₂— C17-1 C17-1 H 2-45 Me 0 3 0 —(CH₂)₂— C17-1 C17-1 H 2-46 Me 0 40 —(CH₂)₂— C17-1 C17-1 H 2-47 Me 1 1 0 —(CH₂)₂— C17-1 C17-1 H 2-48 Me 12 0 —(CH₂)₂— C17-1 C17-1 H 2-49 Me 1 3 0 —(CH₂)₂— C17-1 C17-1 H 2-50 Me2 2 0 —(CH₂)₂— C17-1 C17-1 H 2-51 Me 0 2 0 —(CH₂)₃— C17-1 C17-1 H 2-52Me 0 3 0 —(CH₂)₃— C17-1 C17-1 H 2-53 Me 0 4 0 —(CH₂)₃— C17-1 C17-1 H2-54 Me 1 1 0 —(CH₂)₃— C17-1 C17-1 H 2-55 Me 1 2 0 —(CH₂)₃— C17-1 C17-1H 2-56 Me 1 3 0 —(CH₂)₃— C17-1 C17-1 H 2-57 Me 2 2 0 —(CH₂)₃— C17-1C17-1 H 2-58 Me 1 3 0 — C17-1 C17-1 Me 2-59 Me 2 2 0 — C17-1 C17-1 Me2-60 Me 1 3 0 —CH₂— C17-1 C17-1 Me 2-61 Me 0 2 0 — C17-2 C10 H 2-62 Me 03 0 — C17-2 C10 H 2-63 Me 0 4 0 — C17-2 C10 H 2-64 Me 1 2 0 — C17-2 C10H 2-65 Me 1 3 0 — C17-2 C10 H 2-66 Me 2 2 0 — C17-2 C10 H 2-67 Me 0 2 0—CH₂— C17-2 C10 H 2-68 Me 0 3 0 —CH₂— C17-2 C10 H 2-69 Me 0 4 0 —CH₂—C17-2 C10 H 2-70 Me 1 1 0 —CH₂— C17-2 C10 H 2-71 Me 1 2 0 —CH₂— C17-2C10 H 2-72 Me 1 3 0 —CH₂— C17-2 C10 H 2-73 Me 2 2 0 —CH₂— C17-2 C10 H2-74 Me 0 2 0 —(CH₂)₂— C17-2 C10 H 2-75 Me 0 3 0 —(CH₂)₂— C17-2 C10 H2-76 Me 0 4 0 —(CH₂)₂— C17-2 C10 H 2-77 Me 1 1 0 —(CH₂)₂— C17-2 C10 H2-78 Me 1 2 0 —(CH₂)₂— C17-2 C10 H 2-79 Me 1 3 0 —(CH₂)₂— C17-2 C10 H2-80 Me 2 2 0 —(CH₂)₂— C17-2 C10 H 2-81 Me 0 2 0 —(CH₂)₃— C17-2 C10 H2-82 Me 0 3 0 —(CH₂)₃— C17-2 C10 H 2-83 Me 0 4 0 —(CH₂)₃— C17-2 C10 H2-84 Me 1 1 0 —(CH₂)₃— C17-2 C10 H 2-85 Me 1 2 0 —(CH₂)₃— C17-2 C10 H2-86 Me 1 3 0 —(CH₂)₃— C17-2 C10 H 2-87 Me 2 2 0 —(CH₂)₃— C17-2 C10 H2-88 Me 1 3 0 — C17-2 C10 Me 2-89 Me 2 2 0 — C17-2 C10 Me 2-90 Me 1 3 0—CH₂— C17-2 C10 Me 2-91 Me 0 2 0 — C17-2 C17-2 H 2-92 Me 0 3 0 — C17-2C17-2 H 2-93 Me 0 4 0 — C17-2 C17-2 H 2-94 Me 1 2 0 — C17-2 C17-2 H 2-95Me 1 3 0 — C17-2 C17-2 H 2-96 Me 2 2 0 — C17-2 C17-2 H 2-97 Me 0 2 0—CH₂— C17-2 C17-2 H 2-98 Me 0 3 0 —CH₂— C17-2 C17-2 H 2-99 Me 0 4 0—CH₂— C17-2 C17-2 H 2-100 Me 1 1 0 —CH₂— C17-2 C17-2 H 2-101 Me 1 2 0—CH₂— C17-2 C17-2 H 2-102 Me 1 3 0 —CH₂— C17-2 C17-2 H 2-103 Me 2 2 0—CH₂— C17-2 C17-2 H 2-104 Me 0 2 0 —(CH₂)₂— C17-2 C17-2 H 2-105 Me 0 3 0—(CH₂)₂— C17-2 C17-2 H 2-106 Me 0 4 0 —(CH₂)₂— C17-2 C17-2 H 2-107 Me 11 0 —(CH₂)₂— C17-2 C17-2 H 2-108 Me 1 2 0 —(CH₂)₂— C17-2 C17-2 H 2-109Me 1 3 0 —(CH₂)₂— C17-2 C17-2 H 2-110 Me 2 2 0 —(CH₂)₂— C17-2 C17-2 H2-111 Me 0 2 0 —(CH₂)₃— C17-2 C17-2 H 2-112 Me 0 3 0 —(CH₂)₃— C17-2C17-2 H 2-113 Me 0 4 0 —(CH₂)₃— C17-2 C17-2 H 2-114 Me 1 1 0 —(CH₂)₃—C17-2 C17-2 H 2-115 Me 1 2 0 —(CH₂)₃— C17-2 C17-2 H 2-116 Me 1 3 0—(CH₂)₃— C17-2 C17-2 H 2-117 Me 2 2 0 —(CH₂)₃— C17-2 C17-2 H 2-118 Me 13 0 — C17-2 C17-2 Me 2-119 Me 2 2 0 — C17-2 C17-2 Me 2-120 Me 1 3 0—CH₂— C17-2 C17-2 Me 2-121 Me 0 2 0 — C17-A C10 H 2-122 Me 0 3 0 — C17-AC10 H 2-123 Me 0 4 0 — C17-A C10 H 2-124 Me 1 2 0 — C17-A C10 H 2-125 Me1 3 0 — C17-A C10 H 2-126 Me 2 2 0 — C17-A C10 H 2-127 Me 0 2 0 —CH₂—C17-A C10 H 2-128 Me 0 3 0 —CH₂— C17-A C10 H 2-129 Me 0 4 0 —CH₂— C17-AC10 H 2-130 Me 1 1 0 —CH₂— C17-A C10 H 2-131 Me 1 2 0 —CH₂— C17-A C10 H2-132 Me 1 3 0 —CH₂— C17-A C10 H 2-133 Me 2 2 0 —CH₂— C17-A C10 H 2-134Me 0 2 0 —(CH₂)₂— C17-A C10 H 2-135 Me 0 3 0 —(CH₂)₂— C17-A C10 H 2-136Me 0 4 0 —(CH₂)₂— C17-A C10 H 2-137 Me 1 1 0 —(CH₂)₂— C17-A C10 H 2-138Me 1 2 0 —(CH₂)₂— C17-A C10 H 2-139 Me 1 3 0 —(CH₂)₂— C17-A C10 H 2-140Me 2 2 0 —(CH₂)₂— C17-A C10 H 2-141 Me 0 2 0 —(CH₂)₃— C17-A C10 H 2-142Me 0 3 0 —(CH₂)₃— C17-A C10 H 2-143 Me 0 4 0 —(CH₂)₃— C17-A C10 H 2-144Me 1 1 0 —(CH₂)₃— C17-A C10 H 2-145 Me 1 2 0 —(CH₂)₃— C17-A C10 H 2-146Me 1 3 0 —(CH₂)₃— C17-A C10 H 2-147 Me 2 2 0 —(CH₂)₃— C17-A C10 H 2-148Me 1 3 0 — C17-A C10 Me 2-149 Me 2 2 0 — C17-A C10 Me 2-150 Me 1 3 0—CH₂— C17-A C10 Me 2-151 Me 0 2 0 — C17-A C17-A H 2-152 Me 0 3 0 — C17-AC17-A H 2-153 Me 0 4 0 — C17-A C17-A H 2-154 Me 1 2 0 — C17-A C17-A H2-155 Me 1 3 0 — C17-A C17-A H 2-156 Me 2 2 0 — C17-A C17-A H 2-157 Me 02 0 —CH₂— C17-A C17-A H 2-158 Me 0 3 0 —CH₂— C17-A C17-A H 2-159 Me 0 40 —CH₂— C17-A C17-A H 2-160 Me 1 1 0 —CH₂— C17-A C17-A H 2-161 Me 1 2 0—CH₂— C17-A C17-A H 2-162 Me 1 3 0 —CH₂— C17-A C17-A H 2-163 Me 2 2 0—CH₂— C17-A C17-A H 2-164 Me 0 2 0 —(CH₂)₂— C17-A C17-A H 2-165 Me 0 3 0—(CH₂)₂— C17-A C17-A H 2-166 Me 0 4 0 —(CH₂)₂— C17-A C17-A H 2-167 Me 11 0 —(CH₂)₂— C17-A C17-A H 2-168 Me 1 2 0 —(CH₂)₂— C17-A C17-A H 2-169Me 1 3 0 —(CH₂)₂— C17-A C17-A H 2-170 Me 2 2 0 —(CH₂)₂— C17-A C17-A H2-171 Me 0 2 0 —(CH₂)₃— C17-A C17-A H 2-172 Me 0 3 0 —(CH₂)₃— C17-AC17-A H 2-173 Me 0 4 0 —(CH₂)₃— C17-A C17-A H 2-174 Me 1 1 0 —(CH₂)₃—C17-A C17-A H 2-175 Me 1 2 0 —(CH₂)₃— C17-A C17-A H 2-176 Me 1 3 0—(CH₂)₃— C17-A C17-A H 2-177 Me 2 2 0 —(CH₂)₃— C17-A C17-A H 2-178 Me 13 0 — C17-A C17-A Me 2-179 Me 2 2 0 — C17-A C17-A Me 2-180 Me 1 3 0—CH₂— C17-A C17-A Me 2-181 Me 0 2 0 — C17-H C10 H 2-182 Me 0 3 0 — C17-HC10 H 2-183 Me 0 4 0 — C17-H C10 H 2-184 Me 1 2 0 — C17-H C10 H 2-185 Me1 3 0 — C17-H C10 H 2-186 Me 2 2 0 — C17-H C10 H 2-187 Me 0 2 0 —CH₂—C17-H C10 H 2-188 Me 0 3 0 —CH₂— C17-H C10 H 2-189 Me 0 4 0 —CH₂— C17-HC10 H 2-190 Me 1 1 0 —CH₂— C17-H C10 H 2-191 Me 1 2 0 —CH₂— C17-H C10 H2-192 Me 1 3 0 —CH₂— C17-H C10 H 2-193 Me 2 2 0 —CH₂— C17-H C10 H 2-194Me 0 2 0 —(CH₂)₂— C17-H C10 H 2-195 Me 0 3 0 —(CH₂)₂— C17-H C10 H 2-196Me 0 4 0 —(CH₂)₂— C17-H C10 H 2-197 Me 1 1 0 —(CH₂)₂— C17-H C10 H 2-198Me 1 2 0 —(CH₂)₂— C17-H C10 H 2-199 Me 1 3 0 —(CH₂)₂— C17-H C10 H 2-200Me 2 2 0 —(CH₂)₂— C17-H C10 H 2-201 Me 0 2 0 —(CH₂)₃— C17-H C10 H 2-202Me 0 3 0 —(CH₂)₃— C17-H C10 H 2-203 Me 0 4 0 —(CH₂)₃— C17-H C10 H 2-204Me 1 1 0 —(CH₂)₃— C17-H C10 H 2-205 Me 1 2 0 —(CH₂)₃— C17-H C10 H 2-206Me 1 3 0 —(CH₂)₃— C17-H C10 H 2-207 Me 2 2 0 —(CH₂)₃— C17-H C10 H 2-208Me 1 3 0 — C17-H C10 Me 2-209 Me 2 2 0 — C17-H C10 Me 2-210 Me 1 3 0—CH₂— C17-H C10 Me 2-211 Me 0 2 0 — C17-H C17-H H 2-212 Me 0 3 0 — C17-HC17-H H 2-213 Me 0 4 0 — C17-H C17-H H 2-214 Me 1 2 0 — C17-H C17-H H2-215 Me 1 3 0 — C17-H C17-H H 2-216 Me 2 2 0 — C17-H C17-H H 2-217 Me 02 0 —CH₂— C17-H C17-H H 2-218 Me 0 3 0 —CH₂— C17-H C17-H H 2-219 Me 0 40 —CH₂— C17-H C17-H H 2-220 Me 1 1 0 —CH₂— C17-H C17-H H 2-221 Me 1 2 0—CH₂— C17-H C17-H H 2-222 Me 1 3 0 —CH₂— C17-H C17-H H 2-223 Me 2 2 0—CH₂— C17-H C17-H H 2-224 Me 0 2 0 —(CH₂)₂— C17-H C17-H H 2-225 Me 0 3 0—(CH₂)₂— C17-H C17-H H 2-226 Me 0 4 0 —(CH₂)₂— C17-H C17-H H 2-227 Me 11 0 —(CH₂)₂— C17-H C17-H H 2-228 Me 1 2 0 —(CH₂)₂— C17-H C17-H H 2-229Me 1 3 0 —(CH₂)₂— C17-H C17-H H 2-230 Me 2 2 0 —(CH₂)₂— C17-H C17-H H2-231 Me 0 2 0 —(CH₂)₃— C17-H C17-H H 2-232 Me 0 3 0 —(CH₂)₃— C17-HC17-H H 2-233 Me 0 4 0 —(CH₂)₃— C17-H C17-H H 2-234 Me 1 1 0 —(CH₂)₃—C17-H C17-H H 2-235 Me 1 2 0 —(CH₂)₃— C17-H C17-H H 2-236 Me 1 3 0—(CH₂)₃— C17-H C17-H H 2-237 Me 2 2 0 —(CH₂)₃— C17-H C17-H H 2-238 Me 13 0 — C17-H C17-H Me 2-239 Me 2 2 0 — C17-H C17-H Me 2-240 Me 1 3 0—CH₂— C17-H C17-H Me 2-241 Me 0 2 0 — C17-T C10 H 2-242 Me 0 3 0 — C17-TC10 H 2-243 Me 0 4 0 — C17-T C10 H 2-244 Me 1 2 0 — C17-T C10 H 2-245 Me1 3 0 — C17-T C10 H 2-246 Me 2 2 0 — C17-T C10 H 2-247 Me 0 2 0 —CH₂—C17-T C10 H 2-248 Me 0 3 0 —CH₂— C17-T C10 H 2-249 Me 0 4 0 —CH₂— C17-TC10 H 2-250 Me 1 1 0 —CH₂— C17-T C10 H 2-251 Me 1 2 0 —CH₂— C17-T C10 H2-252 Me 1 3 0 —CH₂— C17-T C10 H 2-253 Me 2 2 0 —CH₂— C17-T C10 H 2-254Me 0 2 0 —(CH₂)₂— C17-T C10 H 2-255 Me 0 3 0 —(CH₂)₂— C17-T C10 H 2-256Me 0 4 0 —(CH₂)₂— C17-T C10 H 2-257 Me 1 1 0 —(CH₂)₂— C17-T C10 H 2-258Me 1 2 0 —(CH₂)₂— C17-T C10 H 2-259 Me 1 3 0 —(CH₂)₂— C17-T C10 H 2-260Me 2 2 0 —(CH₂)₂— C17-T C10 H 2-261 Me 0 2 0 —(CH₂)₃— C17-T C10 H 2-262Me 0 3 0 —(CH₂)₃— C17-T C10 H 2-263 Me 0 4 0 —(CH₂)₃— C17-T C10 H 2-264Me 1 1 0 —(CH₂)₃— C17-T C10 H 2-265 Me 1 2 0 —(CH₂)₃— C17-T C10 H 2-266Me 1 3 0 —(CH₂)₃— C17-T C10 H 2-267 Me 2 2 0 —(CH₂)₃— C17-T C10 H 2-268Me 1 3 0 — C17-T C10 Me 2-269 Me 2 2 0 — C17-T C10 Me 2-270 Me 1 3 0—CH₂— C17-T C10 Me 2-271 Me 0 2 0 — C17-T C17-T H 2-272 Me 0 3 0 — C17-TC17-T H 2-273 Me 0 4 0 — C17-T C17-T H 2-274 Me 1 2 0 — C17-T C17-T H2-275 Me 1 3 0 — C17-T C17-T H 2-276 Me 2 2 0 — C17-T C17-T H 2-277 Me 02 0 —CH₂— C17-T C17-T H 2-278 Me 0 3 0 —CH₂— C17-T C17-T H 2-279 Me 0 40 —CH₂— C17-T C17-T H 2-280 Me 1 1 0 —CH₂— C17-T C17-T H 2-281 Me 1 2 0—CH₂— C17-T C17-T H 2-282 Me 1 3 0 —CH₂— C17-T C17-T H 2-283 Me 2 2 0—CH₂— C17-T C17-T H 2-284 Me 0 2 0 —(CH₂)₂— C17-T C17-T H 2-285 Me 0 3 0—(CH₂)₂— C17-T C17-T H 2-286 Me 0 4 0 —(CH₂)₂— C17-T C17-T H 2-287 Me 11 0 —(CH₂)₂— C17-T C17-T H 2-288 Me 1 2 0 —(CH₂)₂— C17-T C17-T H 2-289Me 1 3 0 —(CH₂)₂— C17-T C17-T H 2-290 Me 2 2 0 —(CH₂)₂— C17-T C17-T H2-291 Me 0 2 0 —(CH₂)₃— C17-T C17-T H 2-292 Me 0 3 0 —(CH₂)₃— C17-TC17-T H 2-293 Me 0 4 0 —(CH₂)₃— C17-T C17-T H 2-294 Me 1 1 0 —(CH₂)₃—C17-T C17-T H 2-295 Me 1 2 0 —(CH₂)₃— C17-T C17-T H 2-296 Me 1 3 0—(CH₂)₃— C17-T C17-T H 2-297 Me 2 2 0 —(CH₂)₃— C17-T C17-T H 2-298 Me 13 0 — C17-T C17-T Me 2-299 Me 2 2 0 — C17-T C17-T Me 2-300 Me 1 3 0—CH₂— C17-T C17-T Me 2-301 Me 0 2 0 — C17-T2 C10 H 2-302 Me 0 3 0 —C17-T2 C10 H 2-303 Me 0 4 0 — C17-T2 C10 H 2-304 Me 1 2 0 — C17-T2 C10 H2-305 Me 1 3 0 — C17-T2 C10 H 2-306 Me 2 2 0 — C17-T2 C10 H 2-307 Me 0 20 —CH₂— C17-T2 C10 H 2-308 Me 0 3 0 —CH₂— C17-T2 C10 H 2-309 Me 0 4 0—CH₂— C17-T2 C10 H 2-310 Me 1 1 0 —CH₂— C17-T2 C10 H 2-311 Me 1 2 0—CH₂— C17-T2 C10 H 2-312 Me 1 3 0 —CH₂— C17-T2 C10 H 2-313 Me 2 2 0—CH₂— C17-T2 C10 H 2-314 Me 0 2 0 —(CH₂)₂— C17-T2 C10 H 2-315 Me 0 3 0—(CH₂)₂— C17-T2 C10 H 2-316 Me 0 4 0 —(CH₂)₂— C17-T2 C10 H 2-317 Me 1 10 —(CH₂)₂— C17-T2 C10 H 2-318 Me 1 2 0 —(CH₂)₂— C17-T2 C10 H 2-319 Me 13 0 —(CH₂)₂— C17-T2 C10 H 2-320 Me 2 2 0 —(CH₂)₂— C17-T2 C10 H 2-321 Me0 2 0 —(CH₂)₃— C17-T2 C10 H 2-322 Me 0 3 0 —(CH₂)₃— C17-T2 C10 H 2-323Me 0 4 0 —(CH₂)₃— C17-T2 C10 H 2-324 Me 1 1 0 —(CH₂)₃— C17-T2 C10 H2-325 Me 1 2 0 —(CH₂)₃— C17-T2 C10 H 2-326 Me 1 3 0 —(CH₂)₃— C17-T2 C10H 2-327 Me 2 2 0 —(CH₂)₃— C17-T2 C10 H 2-328 Me 1 3 0 — C17-T2 C10 Me2-329 Me 2 2 0 — C17-T2 C10 Me 2-330 Me 1 3 0 —CH₂— C17-T2 C10 Me 2-331Me 0 2 0 — C17-T2 C17-T2 H 2-332 Me 0 3 0 — C17-T2 C17-T2 H 2-333 Me 0 40 — C17-T2 C17-T2 H 2-334 Me 1 2 0 — C17-T2 C17-T2 H 2-335 Me 1 3 0 —C17-T2 C17-T2 H 2-336 Me 2 2 0 — C17-T2 C17-T2 H 2-337 Me 0 2 0 —CH₂—C17-T2 C17-T2 H 2-338 Me 0 3 0 —CH₂— C17-T2 C17-T2 H 2-339 Me 0 4 0—CH₂— C17-T2 C17-T2 H 2-340 Me 1 1 0 —CH₂— C17-T2 C17-T2 H 2-341 Me 1 20 —CH₂— C17-T2 C17-T2 H 2-342 Me 1 3 0 —CH₂— C17-T2 C17-T2 H 2-343 Me 22 0 —CH₂— C17-T2 C17-T2 H 2-344 Me 0 2 0 —(CH₂)₂— C17-T2 C17-T2 H 2-345Me 0 3 0 —(CH₂)₂— C17-T2 C17-T2 H 2-346 Me 0 4 0 —(CH₂)₂— C17-T2 C17-T2H 2-347 Me 1 1 0 —(CH₂)₂— C17-T2 C17-T2 H 2-348 Me 1 2 0 —(CH₂)₂— C17-T2C17-T2 H 2-349 Me 1 3 0 —(CH₂)₂— C17-T2 C17-T2 H 2-350 Me 2 2 0 —(CH₂)₂—C17-T2 C17-T2 H 2-351 Me 0 2 0 —(CH₂)₃— C17-T2 C17-T2 H 2-352 Me 0 3 0—(CH₂)₃— C17-T2 C17-T2 H 2-353 Me 0 4 0 —(CH₂)₃— C17-T2 C17-T2 H 2-354Me 1 1 0 —(CH₂)₃— C17-T2 C17-T2 H 2-355 Me 1 2 0 —(CH₂)₃— C17-T2 C17-T2H 2-356 Me 1 3 0 —(CH₂)₃— C17-T2 C17-T2 H 2-357 Me 2 2 0 —(CH₂)₃— C17-T2C17-T2 H 2-358 Me 1 3 0 — C17-T2 C17-T2 Me 2-359 Me 2 2 0 — C17-T2C17-T2 Me 2-360 Me 1 3 0 —CH₂— C17-T2 C17-T2 Me 2-361 Me 0 2 0 — Lin LinH 2-362 Me 0 3 0 — Lin Lin H 2-363 Me 0 4 0 — Lin Lin H 2-364 Me 1 2 0 —Lin Lin H 2-365 Me 1 3 0 — Lin Lin H 2-366 Me 2 2 0 — Lin Lin H 2-367 Me0 2 0 —CH₂— Lin Lin H 2-368 Me 0 3 0 —CH₂— Lin Lin H 2-369 Me 0 4 0—CH₂— Lin Lin H 2-370 Me 1 1 0 —CH₂— Lin Lin H 2-371 Me 1 2 0 —CH₂— LinLin H 2-372 Me 1 3 0 —CH₂— Lin Lin H 2-373 Me 2 2 0 —CH₂— Lin Lin H2-374 Me 0 2 0 —(CH₂)₂— Lin Lin H 2-375 Me 0 3 0 —(CH₂)₂— Lin Lin H2-376 Me 0 4 0 —(CH₂)₂— Lin Lin H 2-377 Me 1 1 0 —(CH₂)₂— Lin Lin H2-378 Me 1 2 0 —(CH₂)₂— Lin Lin H 2-379 Me 1 3 0 —(CH₂)₂— Lin Lin H2-380 Me 2 2 0 —(CH₂)₂— Lin Lin H 2-381 Me 0 2 0 —(CH₂)₃— Lin Lin H2-382 Me 0 3 0 —(CH₂)₃— Lin Lin H 2-383 Me 0 4 0 —(CH₂)₃— Lin Lin H2-384 Me 1 1 0 —(CH₂)₃— Lin Lin H 2-385 Me 1 2 0 —(CH₂)₃— Lin Lin H2-386 Me 1 3 0 —(CH₂)₃— Lin Lin H 2-387 Me 2 2 0 —(CH₂)₃— Lin Lin H2-388 Me 1 3 0 — Lin Lin Me 2-389 Me 2 2 0 — Lin Lin Me 2-390 Me 1 3 0—CH₂— Lin Lin Me 2-391 Me 0 2 0 — C16 C10 H 2-392 Me 0 3 0 — C16 C10 H2-393 Me 0 4 0 — C16 C10 H 2-394 Me 1 2 0 — C16 C10 H 2-395 Me 1 3 0 —C16 C10 H 2-396 Me 2 2 0 — C16 C10 H 2-397 Me 0 2 0 —CH₂— C16 C10 H2-398 Me 0 3 0 —CH₂— C16 C10 H 2-399 Me 0 4 0 —CH₂— C16 C10 H 2-400 Me 11 0 —CH₂— C16 C10 H 2-401 Me 1 2 0 —CH₂— C16 C10 H 2-402 Me 1 3 0 —CH₂—C16 C10 H 2-403 Me 2 2 0 —CH₂— C16 C10 H 2-404 Me 0 2 0 —(CH₂)₂— C16 C10H 2-405 Me 0 3 0 —(CH₂)₂— C16 C10 H 2-406 Me 0 4 0 —(CH₂)₂— C16 C10 H2-407 Me 1 1 0 —(CH₂)₂— C16 C10 H 2-408 Me 1 2 0 —(CH₂)₂— C16 C10 H2-409 Me 1 3 0 —(CH₂)₂— C16 C10 H 2-410 Me 2 2 0 —(CH₂)₂— C16 C10 H2-411 Me 0 2 0 —(CH₂)₃— C16 C10 H 2-412 Me 0 3 0 —(CH₂)₃— C16 C10 H2-413 Me 0 4 0 —(CH₂)₃— C16 C10 H 2-414 Me 1 1 0 —(CH₂)₃— C16 C10 H2-415 Me 1 2 0 —(CH₂)₃— C16 C10 H 2-416 Me 1 3 0 —(CH₂)₃— C16 C10 H2-417 Me 2 2 0 —(CH₂)₃— C16 C10 H 2-418 Me 1 3 0 — C16 C10 Me 2-419 Me 22 0 — C16 C10 Me 2-420 Me 1 3 0 —CH₂— C16 C10 Me 2-421 Me 0 2 0 — C16C16 H 2-422 Me 0 3 0 — C16 C16 H 2-423 Me 0 4 0 — C16 C16 H 2-424 Me 1 20 — C16 C16 H 2-425 Me 1 3 0 — C16 C16 H 2-426 Me 2 2 0 — C16 C16 H2-427 Me 0 2 0 —CH₂— C16 C16 H 2-428 Me 0 3 0 —CH₂— C16 C16 H 2-429 Me 04 0 —CH₂— C16 C16 H 2-430 Me 1 1 0 —CH₂— C16 C16 H 2-431 Me 1 2 0 —CH₂—C16 C16 H 2-432 Me 1 3 0 —CH₂— C16 C16 H 2-433 Me 2 2 0 —CH₂— C16 C16 H2-434 Me 0 2 0 —(CH₂)₂— C16 C16 H 2-435 Me 0 3 0 —(CH₂)₂— C16 C16 H2-436 Me 0 4 0 —(CH₂)₂— C16 C16 H 2-437 Me 1 1 0 —(CH₂)₂— C16 C16 H2-438 Me 1 2 0 —(CH₂)₂— C16 C16 H 2-439 Me 1 3 0 —(CH₂)₂— C16 C16 H2-440 Me 2 2 0 —(CH₂)₂— C16 C16 H 2-441 Me 0 2 0 —(CH₂)₃— C16 C16 H2-442 Me 0 3 0 —(CH₂)₃— C16 C16 H 2-443 Me 0 4 0 —(CH₂)₃— C16 C16 H2-444 Me 1 1 0 —(CH₂)₃— C16 C16 H 2-445 Me 1 2 0 —(CH₂)₃— C16 C16 H2-446 Me 1 3 0 —(CH₂)₃— C16 C16 H 2-447 Me 2 2 0 —(CH₂)₃— C16 C16 H2-448 Me 1 3 0 — C16 C16 Me 2-449 Me 2 2 0 — C16 C16 Me 2-450 Me 1 3 0—CH₂— C16 C16 Me 2-451 Me 0 2 0 — C17 C10 H 2-452 Me 0 3 0 — C17 C10 H2-453 Me 0 4 0 — C17 C10 H 2-454 Me 1 2 0 — C17 C10 H 2-455 Me 1 3 0 —C17 C10 H 2-456 Me 2 2 0 — C17 C10 H 2-457 Me 0 2 0 —CH₂— C17 C10 H2-458 Me 0 3 0 —CH₂— C17 C10 H 2-459 Me 0 4 0 —CH₂— C17 C10 H 2-460 Me 11 0 —CH₂— C17 C10 H 2-461 Me 1 2 0 —CH₂— C17 C10 H 2-462 Me 1 3 0 —CH₂—C17 C10 H 2-463 Me 2 2 0 —CH₂— C17 C10 H 2-464 Me 0 2 0 —(CH₂)₂— C17 C10H 2-465 Me 0 3 0 —(CH₂)₂— C17 C10 H 2-466 Me 0 4 0 —(CH₂)₂— C17 C10 H2-467 Me 1 1 0 —(CH₂)₂— C17 C10 H 2-468 Me 1 2 0 —(CH₂)₂— C17 C10 H2-469 Me 1 3 0 —(CH₂)₂— C17 C10 H 2-470 Me 2 2 0 —(CH₂)₂— C17 C10 H2-471 Me 0 2 0 —(CH₂)₃— C17 C10 H 2-472 Me 0 3 0 —(CH₂)₃— C17 C10 H2-473 Me 0 4 0 —(CH₂)₃— C17 C10 H 2-474 Me 1 1 0 —(CH₂)₃— C17 C10 H2-475 Me 1 2 0 —(CH₂)₃— C17 C10 H 2-476 Me 1 3 0 —(CH₂)₃— C17 C10 H2-477 Me 2 2 0 —(CH₂)₃— C17 C10 H 2-478 Me 1 3 0 — C17 C10 Me 2-479 Me 22 0 — C17 C10 Me 2-480 Me 1 3 0 —CH₂— C17 C10 Me 2-481 Me 0 2 0 — C17C17 H 2-482 Me 0 3 0 — C17 C17 H 2-483 Me 0 4 0 — C17 C17 H 2-484 Me 1 20 — C17 C17 H 2-485 Me 1 3 0 — C17 C17 H 2-486 Me 2 2 0 — C17 C17 H2-487 Me 0 2 0 —CH₂— C17 C17 H 2-488 Me 0 3 0 —CH₂— C17 C17 H 2-489 Me 04 0 —CH₂— C17 C17 H 2-490 Me 1 1 0 —CH₂— C17 C17 H 2-491 Me 1 2 0 —CH₂—C17 C17 H 2-492 Me 1 3 0 —CH₂— C17 C17 H 2-493 Me 2 2 0 —CH₂— C17 C17 H2-494 Me 0 2 0 —(CH₂)₂— C17 C17 H 2-495 Me 0 3 0 —(CH₂)₂— C17 C17 H2-496 Me 0 4 0 —(CH₂)₂— C17 C17 H 2-497 Me 1 1 0 —(CH₂)₂— C17 C17 H2-498 Me 1 2 0 —(CH₂)₂— C17 C17 H 2-499 Me 1 3 0 —(CH₂)₂— C17 C17 H2-500 Me 2 2 0 —(CH₂)₂— C17 C17 H 2-501 Me 0 2 0 —(CH₂)₃— C17 C17 H2-502 Me 0 3 0 —(CH₂)₃— C17 C17 H 2-503 Me 0 4 0 —(CH₂)₃— C17 C17 H2-504 Me 1 1 0 —(CH₂)₃— C17 C17 H 2-505 Me 1 2 0 —(CH₂)₃— C17 C17 H2-506 Me 1 3 0 —(CH₂)₃— C17 C17 H 2-507 Me 2 2 0 —(CH₂)₃— C17 C17 H2-508 Me 1 3 0 — C17 C17 Me 2-509 Me 2 2 0 — C17 C17 Me 2-510 Me 1 3 0—CH₂— C17 C17 Me 2-511 Me 0 2 0 — C18 C10 H 2-512 Me 0 3 0 — C18 C10 H2-513 Me 0 4 0 — C18 C10 H 2-514 Me 1 2 0 — C18 C10 H 2-515 Me 1 3 0 —C18 C10 H 2-516 Me 2 2 0 — C18 C10 H 2-517 Me 0 2 0 —CH₂— C18 C10 H2-518 Me 0 3 0 —CH₂— C18 C10 H 2-519 Me 0 4 0 —CH₂— C18 C10 H 2-520 Me 11 0 —CH₂— C18 C10 H 2-521 Me 1 2 0 —CH₂— C18 C10 H 2-522 Me 1 3 0 —CH₂—C18 C10 H 2-523 Me 2 2 0 —CH₂— C18 C10 H 2-524 Me 0 2 0 —(CH₂)₂— C18 C10H 2-525 Me 0 3 0 —(CH₂)₂— C18 C10 H 2-526 Me 0 4 0 —(CH₂)₂— C18 C10 H2-527 Me 1 1 0 —(CH₂)₂— C18 C10 H 2-528 Me 1 2 0 —(CH₂)₂— C18 C10 H2-529 Me 1 3 0 —(CH₂)₂— C18 C10 H 2-530 Me 2 2 0 —(CH₂)₂— C18 C10 H2-531 Me 0 2 0 —(CH₂)₃— C18 C10 H 2-532 Me 0 3 0 —(CH₂)₃— C18 C10 H2-533 Me 0 4 0 —(CH₂)₃— C18 C10 H 2-534 Me 1 1 0 —(CH₂)₃— C18 C10 H2-535 Me 1 2 0 —(CH₂)₃— C18 C10 H 2-536 Me 1 3 0 —(CH₂)₃— C18 C10 H2-537 Me 2 2 0 —(CH₂)₃— C18 C10 H 2-538 Me 1 3 0 — C18 C10 Me 2-539 Me 22 0 — C18 C10 Me 2-540 Me 1 3 0 —CH₂— C18 C10 Me 2-541 Me 0 2 0 — C18C18 H 2-542 Me 0 3 0 — C18 C18 H 2-543 Me 0 4 0 — C18 C18 H 2-544 Me 1 20 — C18 C18 H 2-545 Me 1 3 0 — C18 C18 H 2-546 Me 2 2 0 — C18 C18 H2-547 Me 0 2 0 —CH₂— C18 C18 H 2-548 Me 0 3 0 —CH₂— C18 C18 H 2-549 Me 04 0 —CH₂— C18 C18 H 2-550 Me 1 1 0 —CH₂— C18 C18 H 2-551 Me 1 2 0 —CH₂—C18 C18 H 2-552 Me 1 3 0 —CH₂— C18 C18 H 2-553 Me 2 2 0 —CH₂— C18 C18 H2-554 Me 0 2 0 —(CH₂)₂— C18 C18 H 2-555 Me 0 3 0 —(CH₂)₂— C18 C18 H2-556 Me 0 4 0 —(CH₂)₂— C18 C18 H 2-557 Me 1 1 0 —(CH₂)₂— C18 C18 H2-558 Me 1 2 0 —(CH₂)₂— C18 C18 H 2-559 Me 1 3 0 —(CH₂)₂— C18 C18 H2-560 Me 2 2 0 —(CH₂)₂— C18 C18 H 2-561 Me 0 2 0 —(CH₂)₃— C18 C18 H2-562 Me 0 3 0 —(CH₂)₃— C18 C18 H 2-563 Me 0 4 0 —(CH₂)₃— C18 C18 H2-564 Me 1 1 0 —(CH₂)₃— C18 C18 H 2-565 Me 1 2 0 —(CH₂)₃— C18 C18 H2-566 Me 1 3 0 —(CH₂)₃— C18 C18 H 2-567 Me 2 2 0 —(CH₂)₃— C18 C18 H2-568 Me 1 3 0 — C18 C18 Me 2-569 Me 2 2 0 — C18 C18 Me 2-570 Me 1 3 0—CH₂— C18 C18 Me1-3. Method for Producing Cationic Lipid

The cationic lipid of the present invention can be produced by theapplication of various synthesis methods known in the art. The synthesismethods known in the art include those described in “ComprehensiveOrganic Transformations (2nd ed.)”, Wiley-VCH, 1999, “ComprehensiveOrganic Synthesis”, Pergamon Press, 1991, etc. Depending on the type ofthe functional group used, the protection of a starting material or anintermediate with a suitable protective group, or reaction afterreplacement with a functional group that can be easily converted to thefunctional group of interest may be effective for the production. Such afunctional group includes a hydroxy group, a carboxyl group, an aminogroup, multiple bonds, and the like. The protection and deprotection ofthe functional group or derivatization to the functional group can becarried out by a method known in the art. The method known in the artincludes “Protective Groups in Organic Synthesis (4th ed.)”,Wiley-Interscience, 2006, etc.

The method for producing the cationic lipid (I) of the presentinvention, which is summarized in FIGS. 1 to 3, will be shown below.However, the production method is not limited to the methods describedbelow.

In the formulas, G¹, G², G³, G⁴, and G⁵ each independently represent ahydrogen atom, a substituent selected from substituent group β, or asubstituent that can serve as a synthetic-chemically acceptableprotected form or precursor for inducing any substituent of substituentgroup β.

p¹ and p² each independently represent an integer of 0 to 21.

G⁶, G⁷, G¹⁰, G¹¹, and G¹² each independently represent a C₁₀-C₂₄ alkylgroup optionally having one or more substituents selected fromsubstituent group β, a C₁₀-C₂₄ alkenyl group optionally having one ormore substituents selected from substituent group β, a C₃-C₂₄ alkynylgroup optionally having one or more substituents selected fromsubstituent group β, a (C₁-C₁₀ alkyl)-(Q)_(k)-(C₁-C₁₀ alkyl) groupoptionally having one or more substituents selected from substituentgroup β, or a substituent that can serve as a synthetic-chemicallyacceptable protected form or precursor for inducing any of thesesubstituents.

G⁸ represents a substituent represented by the following structuralformula, or a substituent that can serve as a synthetic-chemicallyacceptable protected form or precursor for inducing the substituent:

G⁹ is not particularly limited as long as it can be generally used as aprotective group for a carboxyl group. Examples thereof include a methylgroup, an ethyl group, a propyl group, an isopropyl group, a butylgroup, an isobutyl group, a sec-butyl group, a tert-butyl group, abenzyl group, a p-nitrobenzyl group, an o-nitrobenzyl group, and ap-methoxybenzyl group.

1-3-1. Method A

Method A is summarized in FIG. 1.

1-3-1-1. Step A-1

Step A-1 is the step of producing an internal alkyne compound (A3)having a hydroxy group from a terminal alkyne compound (A1) having ahydroxy group and an alkyl compound (A2) having a leaving substituent.

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,sulfoxide, and halogenated hydrocarbon solvents. Aprotic polar solventssuch as amide and sulfoxide solvents are preferred, andN,N-dimethylformamide (DMF) is more preferred.

Examples of the reagent used include transition metal compounds. Coppercompounds are preferred, and copper(I) iodide is more preferred.

Two or more of inorganic salts, inorganic bases, alkali metal alkoxides,alkaline earth metal alkoxides, organic bases, and the like are used asadditives. Inorganic salts and inorganic bases are preferred, and sodiumiodide and potassium carbonate are more preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-1-2. Step A-2

Step A-2 is the step of producing an internal alkyne compound (A4)having a formyl group from the internal alkyne compound (A3) having ahydroxy group.

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include sulfoxide solvents. Dimethyl sulfoxide(DMSO) is preferred.

Examples of the reagent used include sulfur trioxide-pyridine complexesand oxalic acid chloride.

Inorganic bases and organic bases are used as additives. Organic basesare preferred, and triethylamine is more preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-1-3. Step A-3

Step A-3 is the step of producing an internal alkyne compound (A5)having a carboxyl group from the internal alkyne compound (A4) having aformyl group.

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include alcohol, amine, and aqueous solvents.Alcohol solvents are preferred, and tert-butyl alcohol is morepreferred.

Examples of the reagent used include inorganic oxidizing agents such assodium chlorite.

Inorganic salts, inorganic bases, alkali metal alkoxides, and alkalineearth metal alkoxides are used as additives. Phosphates are preferred,and sodium dihydrogen phosphates are more preferred.

The additive further used is, for example, 2-methyl-2-butene.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2. Method B

Method B is summarized in FIG. 2.

1-3-2-1. Step B-1

Step B-1 is the step of producing a N-methoxy carboxylic acid amide (B2)from a carboxylic acid (B1).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,sulfoxide, and halogenated hydrocarbon solvents. Halogenated hydrocarbonsolvents are preferred, and dichloromethane is more preferred.

Examples of the secondary material used includeN,O-dimethylhydroxylamine hydrochloride.

Examples of the reagent used include dehydrative condensation agentssuch as carbodiimides. 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimidehydrochloride is preferred.

Inorganic bases, alkali metal alkoxides, alkaline earth metal alkoxides,and organic bases are used as additives. Organic bases are preferred,and triethylamine is more preferred. The additive further used is aN-hydroxy heterocyclic compound as a dehydrative condensation activator.1-Hydroxybenzimidazole hydrate is preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-2. Step B-2

Step B-2 is the step of producing a ketone (B3) from the N-methoxycarboxylic acid amide (B2).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,and sulfoxide solvents. Ether solvents such as diethyl ether andtetrahydrofuran are preferred.

Examples of the reagent used include Grignard reagents, alkyllithiumreagents, and alkylzinc reagents. Grignard reagents and alkyllithiumreagents are preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 100° C., preferably -30° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-3. Step B-3

Step B-3 is the step of producing an alcohol (B4) from the ketone (B3).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include ether, ester, amide, nitrile,sulfoxide, alcohol, amine, and aqueous solvents. Protonic solvents suchas alcohol and aqueous solvents are preferred, and an aqueous solutionof methanol or ethanol is more preferred.

Examples of the reagent used include boron reagents and aluminumreagents generally used as reducing agents. Sodium borohydride ispreferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-4. Step B-4

Step B-4 is the step of producing an alcohol (B4′) having a double bondin the molecule from the alcohol (B4) having a triple bond in themolecule.

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, alcohol,amine, and aqueous solvents. Protonic solvents such as alcohol andaqueous solvents are preferred, and methanol or ethanol is morepreferred.

Examples of the secondary material used include reducing agents ashydrogen gas or hydrogen donors. Hydrogen, sodium borohydride, and thelike are preferred. These secondary materials are used alone or incombination of two or more thereof.

Examples of the catalyst used include transition metal compounds.Supported palladium metals, nickel compounds, and cobalt compounds arepreferred, and polyethyleneimine-supported palladium and nickel(II)acetate tetrahydrate are more preferred.

In the case of using a nickel compound, a cobalt compound, or the likeas the catalyst, organic bases are used as additives. Ethylenediamine ispreferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 80° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-5. Step B-5

Step B-5 is the step of producing a carbonate (B5) from the alcohol(B4).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,sulfoxide, halogenated hydrocarbon, and amine solvents. Low polarsolvents such as aromatic and halogenated hydrocarbon solvents arepreferred, and toluene and dichloromethane are more preferred.

Examples of the secondary material used include alcohols and activecarbonates such as p-nitrophenyl-substituted carbonate.

In the case of using an alcohol as the secondary material, examples ofthe reagent used include phosgene equivalents. Diphosgene andtriphosgene are preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 100° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-6. Step B-6

Step B-6 is the step of producing a carbonate (B5′) having a double bondin the molecule from the carbonate (B5) having a triple bond in themolecule.

This step can be carried out in the same way as in step B-4.

1-3-2-7. Step B-7

Step B-7 is the step of producing an aldehyde (B7) from the alcohol(B6).

Various general oxidation reactions may be used. In the case of using asulfoxide as an oxidizing agent, the solvent used is preferably dimethylsulfoxide.

Examples of the reagent used include oxalyl chloride, trifluoroaceticacid, dicyclohexylcarbodiimide, and sulfur trioxide-pyridine complexes.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 80° C., preferably 0° C. to 60° C.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-2-8. Step B-8

Step B-8 is the step of producing an alcohol (B4) from the aldehyde(B7).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,and sulfoxide solvents. Ether solvents such as diethyl ether andtetrahydrofuran are preferred.

Examples of the reagent used include Grignard reagents, alkyllithiumreagents, and alkylzinc reagents. Grignard reagents and alkyllithiumreagents are preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 100° C., preferably -30° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3. Method C

Method C is summarized in FIG. 3.

1-3-3-1. Step C-1

Step C-1 is the step of producing a monosubstituted malonate (C2) froman unsubstituted malonate (C1).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,sulfoxide, and alcohol solvents. Aromatic solvents such as toluene andxylene are preferred.

Examples of the secondary material used include alkyl halides and alkylsulfonates.

Examples of the reagent used include inorganic bases, alkali metalalkoxides, alkaline earth metal alkoxides, and organic bases. Sodiumhydride and sodium methoxide are preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 200° C., preferably 50° C. to 150° C.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3-2. Step C-2

Step C-2 is the step of producing an ester (C3) from the monosubstitutedmalonate (C2).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ester, amide, nitrile,sulfoxide, and aqueous solvents. Aprotic polar solvents such as amideand sulfoxide solvents are preferred, and dimethyl sulfoxide (DMSO) ismore preferred.

Examples of the reagent used include inorganic salts and inorganicbases. Lithium chloride and sodium cyanide are preferred.

Examples of the additive used include water.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 80° C.to 200° C., preferably 120° C. to 180° C.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3-3. Step C-3

Step C-3 is the step of producing a ketoester (C4) from the ester (C3).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic, ether, ester, amide, nitrile,and sulfoxide solvents. Aromatic solvents are preferred, and xylenes aremore preferred.

Examples of the secondary material used include various organic acidesters having a structure condensed with the same alcohol as thestarting material.

Examples of the reagent used include inorganic bases, alkali metalalkoxides, alkaline earth metal alkoxides, and organic bases. Sodiumhydride is preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 50° C.to 200° C., preferably 120° C. to 180° C.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3-4. Step C-4

Step C-4 is the step of producing a ketone (C5; compound correspondingto B3) from the ketoester (C4).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include ether, ester, amide, nitrile,sulfoxide, alcohol, and aqueous solvents. Protonic solvents such asalcohol and aqueous solvents are preferred, and an aqueous solution ofmethanol or ethanol is more preferred.

Examples of the reagent used include inorganic bases, alkali metalalkoxides, and alkaline earth metal alkoxides. Sodium hydroxide andlithium hydroxide are preferred.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually 0° C.to 120° C., preferably 50° C. to 100° C.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3-5. Step C-5

Step C-5 is the step of producing a disubstituted malonate (C6) from themonosubstituted malonate (C2).

This step can be carried out in the same way as in step C-1.

1-3-3-6. Step C-6

Step C-6 is the step of producing an ester (C7) from the disubstitutedmalonate (C6).

This step can be carried out in the same way as in step C-2.

1-3-3-7. Step C-7

Step C-7 is the step of producing a 2-substituted alcohol (C8) from theester (C7).

The solvent used is not particularly limited as long as the solvent doesnot inhibit the reaction and can dissolve starting materials to someextent. Examples thereof include aromatic and ether solvents.Tetrahydrofuran is preferred.

Examples of the reagent used include general reducing agents such aslithium aluminum hydride.

The reaction temperature differs depending on the types of the startingmaterials, the solvent, the reagent, and the like and is usually −78° C.to 80° C., preferably 0° C. to room temperature.

After completion of the reaction, the compound of interest of thisreaction is collected from the reaction mixture according to a standardmethod. The compound of interest is obtained, for example, by:appropriately neutralizing the reaction mixture, or removing insolublematter, if any, by filtration; then adding water and a water-immiscibleorganic solvent such as hexane or ethyl acetate; after washing withwater, separating the organic layer containing the compound of interest;drying the organic layer over anhydrous magnesium sulfate, anhydroussodium sulfate, anhydrous sodium bicarbonate, or the like; and thendistilling off the solvent. The obtained compound of interest is furtherpurified, if necessary, by a standard method, for example,recrystallization, reprecipitation, or chromatography.

1-3-3-8. Step C-8

Step C-8 is the step of producing a carbonate (C9) from the2-substituted alcohol (C8).

This step can be carried out in the same way as in step B-5.

2. Lipid Particle

The lipid particle in the present specification includes a compositionhaving any structure selected from a liposome, a lipid aggregate inwhich lipids are aggregated, and a micelle. The structure of the lipidparticle is not limited to them as long as the lipid particle is acomposition containing a lipid. The liposome has a lipid bilayerstructure and has an aqueous phase in the inside. The liposome isclassified as a multilamellar liposome, which has a multilayeredstructure of lipid bilayers, or as a unilamellar liposome, which has onebilayer. The liposome of the present invention includes both suchliposomes.

The “lipid particle” of the present invention includes any compositionselected from the following (a) to (c):

(a) a composition comprising a cationic lipid and a lipid reducingaggregation during lipid particle formation,

(b) a composition comprising a cationic lipid, a lipid reducingaggregation during lipid particle formation, and a sterol, and

(c) a composition comprising a cationic lipid, a lipid reducingaggregation during lipid particle formation, a sterol, and anamphipathic lipid.

In this context, the cationic lipid is one or two or more of variouscationic lipids described in the preceding paragraph “1. Cationiclipid”. Specific examples thereof can include one or two or more of thecompounds described in Table 1 or 2.

Examples of the amphipathic lipid can include one or two or more ofthose described below in the paragraph “2-1. Amphipathic lipid”.

Examples of the sterol can include one or two or more of those describedbelow in the paragraph “2-2. Sterol”.

Examples of the lipid reducing aggregation during lipid particleformation can include one or two or more of those described below in theparagraph “2-3. Lipid reducing aggregation during lipid particleformation”.

2-1. Amphipathic Lipid

In the present specification, the “amphipathic lipid” refers to a lipidhaving affinity for both polar and nonpolar solvents.

Examples of the amphipathic lipid can include lipids described in“Liposomes: from physics to applications”, Chapter 1. Chemistry oflipids and liposomes (published by Elsevier B.V. in 1993, author: D. D.Lasic), etc. The amphipathic lipid includes, for example, phospholipids,glycolipids, aminolipids, sphingolipids, glycols, and saturated orunsaturated fatty acids, though the amphipathic lipid of the presentinvention is not limited to them. Specific examples thereof aredescribed in the paragraphs 2-1-1 to 2-1-3.

2-1-1. Phospholipids

The phospholipids are broadly divided into glycerophospholipids andsphingophospholipids. Typical examples of the glycerophospholipidsinclude phosphatidyl cholines (PC), phosphatidyl serines (PS),phosphatidyl inositols (PI), phosphatidyl glycerols (PG), phosphatidylethanolamines (PE), and phosphatidic acids (PA). On the other hand,typical examples of the sphingophospholipids include sphingomyelin (SM).For example, lipids described in the following (a) to (g) can be listed.

(a) Phosphatidylcholines

Specific examples of the phosphatidylcholines can includedipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dimyristoylphosphatidylcholine (DMPC),dioleoylphosphatidylcholine (DOPC), dilauroylphosphatidylcholine (DLPC),didecanoylphosphatidylcholine (DDPC), dioctanoylphosphatidylcholine(DOPC), dihexanoylphosphatidylcholine (DHPC),dibutyrylphosphatidylcholine (DBPC), dielaidoylphosphatidylcholine(DEPC), dilinoleoylphosphatidylcholine,diarachidonoylphosphatidylcholine, diicosenoylphosphatidylcholine,diheptanoylphosphatidylcholine, dicaproylphosphatidylcholine,diheptadecanoylphosphatidylcholine, dibehenoylphosphatidylcholine,eleostearoylphosphatidylcholine, hydrogenated egg phosphatidylcholine(HEPC), hydrogenated soybean phosphatidylcholine (HSPC),1-palmitoyl-2-arachidonoylphosphatidylcholine,1-palmitoyl-2-oleoylphosphatidylcholine (POPC),1-palmitoyl-2-linoleoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine,1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine,1-myristoyl-2-palmitoylphosphatidylcholine,1-myristoyl-2-stearoylphosphatidylcholine,di-0-hexadecylphosphatidylcholine, trans-dielaidoylphosphatidylcholine,dipalmitelaidoyl-phosphatidylcholine,n-octadecyl-2-methylphosphatidylcholine, n-octadecylphosphatidylcholine,1-laurylpropanedio1-3-phosphocholine,erythro-N-lignoceroylsphingophosphatidylcholine, andpalmitoyl-(9-cis-octadecenoyl)-3-sn-phosphatidylcholine. Preferredexamples thereof can include DSPC, DPPC, and DMPC.

(b) Phosphatidylserines

Specific examples of the phosphatidylserines includedistearoylphosphatidylserine (DSPS), dimyristoylphosphatidylserine(DMPS), dilauroylphosphatidylserine (DLPS),dipalmitoylphosphatidylserine (DPPS), dioleoylphosphatidylserine (DOPS),lysophosphatidylserine, eleostearoylphosphatidylserine, and1,2-di-(9-cis-octadecenoyl)-3-sn-phosphatidylserine.

(c) Phosphatidylinositols

Specific examples of the phosphatidylinositols includedipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI), and dilauroylphosphatidylinositol (DLPI).

(d) Phosphatidylglycerols

Specific examples of the phosphatidylglycerols includedipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol(DSPG), dioleoylphosphatidylglycerol (DOPG),dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol(DMPG), lysophosphatidylglycerol, hydrogenated soybeanphosphatidylglycerol (HSPG), hydrogenated egg phosphatidylglycerol(HEPG), and cardiolipin (diphosphatidylglycerol).

(e) Phosphatidylethanolamines

Specific examples of the phosphatidylethanolamines includedipalmitoylphosphatidylethanolamine (DPPE),distearoylphosphatidylethanolamine (DSPE),dioleoylphosphatidylethanolamine (DOPE),dilauroylphosphatidylethanolamine (DLPE),dimyristoylphosphatidylethanolamine (DMPE),didecanoylphosphatidylethanolamine (DDPE),N-glutarylphosphatidylethanolamine (NGPE), lysophosphatidylethanolamine,N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-1,2-dioleoyl-sn-phosphatidylethanolamine,eleostearoylphosphatidylethanolamine,N-succinyldioleoylphosphatidylethanolamine, and1-hexadecyl-2-palmitoylglycerophosphatidylethanolamine Preferredexamples thereof can include DOPE.

(f) Phosphatidic Acids

Specific examples of the phosphatidic acids includedipalmitoylphosphatidic acid (DPPA), distearoylphosphatidic acid (DSPA),dimyristoylphosphatidic acid (DMPA), and dioleylphosphatidic acid(DOPA).

(g) Sphingophospholipids

Specific examples of the sphingophospholipids include sphingomyelin(SM), dipalmitoylsphingomyelin, distearoylsphingomyelin, ceramideciliatine, ceramide phosphorylethanolamine, and ceramidephosphorylglycerol. Preferred examples thereof can include SM.

2-1-2. Glycolipids

The glycolipids are broadly divided into glyceroglycolipids andsphingoglycolipids. For example, lipids described in the following (a)or (b) can be listed.

(a) Glyceroglycolipids

Specific examples of the glyceroglycolipids include diglycosyldiglyceride, glycosyl diglyceride, digalactosyl diglyceride, galactosyldiglyceride, sulfoxyribosyl diglyceride, (1,3)-D-mannosyl(1,3)diglyceride, digalactosyl glyceride, digalactosyl dilauroylglyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoylglyceride, digalactosyl distearoyl glyceride, galactosyl glyceride,galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride,galactosyl dipalmitoyl glyceride, galactosyl distearoyl glyceride, anddigalactosyl diacyl glycerol.

(b) Sphingoglycolipids

Specific examples of the sphingoglycolipids can include ceramide(cerebroside), galactosyl ceramide, lactosyl ceramide, digalactosylceramide, ganglioside GM1, ganglioside GM2, ganglioside GM3, sulfatide,ceramide oligohexoside, and globoside.

2-1-3. Saturated or Unsaturated Fatty Acids

Specific examples of the saturated fatty acids and the unsaturated fattyacids used include saturated or unsaturated fatty acids each having 5 to30 carbon atoms, such as caprylic acid, pelargonic acid, capric acid,undecylenic acid, lauric acid, tridecylenic acid, myristic acid,pentadecylenic acid, palmitic acid, margaric acid, stearic acid,nonadecylenic acid, arachidic acid, dodecenoic acid, tetradecenoic acid,oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid,and docosapentaenoic acid.

2-2. Sterol

Specific examples of the sterol can include cholesterol, cholesterolsuccinic acid, dihydrocholesterol, lanosterol, dihydrolanosterol,desmosterol, stigmasterol, sitosterol, campesterol, brassicasterol,zymosterol, ergosterol, fucosterol, 22-ketosterol, 20-hydroxysterol,7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol,25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol,epicholesterol, dehydroergosterol, cholesterol sulfate, cholesterolhemisuccinate, cholesterol phthalate, cholesterol phosphate, cholesterolvalerate, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol,cholesterol acetate, cholesteryl oleate, cholesteryl linoleate,cholesteryl myristate, cholesteryl palmitate, cholesteryl arachidate,coprostanol, cholesterol ester, cholesteryl phosphorylcholine, and3,6,9-trioxaoctan-1-ol-cholesteryl-3e-ol. Preferred examples thereof caninclude cholesterol and cholesterol hemisuccinate, more preferablycholesterol.

2-3. Lipid Reducing Aggregation During Lipid Particle Formation

A lipid bound with a nonionic water-soluble polymer can be used as thelipid reducing aggregation during lipid particle formation.

The nonionic water-soluble polymer refers to a polymer having nodissociable group at a site other than the end in an aqueous medium suchas water or a buffer solution, or a polymer derived from the polymersuch that its end is alkoxy. Examples of such a nonionic water-solublepolymer can include:

(1) a nonionic vinyl polymer having, as a constituent, a monomer unitsuch as vinyl alcohol, methyl vinyl ether, vinylpyrrolidone, vinyloxazolidone, vinyl methyl oxazolidone, 2-vinylpyridine, 4-vinylpyridine,N-vinylsuccinimide, N-vinylformamide, N-vinyl-N-methylformamide,N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethylmethacrylate, acrylamide, methacrylamide, N,N-dimethylacrylamide,N-isopropylacrylamide, diacetoneacrylamide, methylolacrylamide,acryloylmorpholine, acryloylpyrrolidine, acryloylpiperidine, styrene,chloromethylstyrene, bromomethylstyrene, vinyl acetate, methylmethacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate,n-propyl cyanoacrylate, isopropyl cyanoacrylate, n-butyl cyanoacrylate,isobutyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate,ethyl vinyl ether, n-propyl vinyl ether, isopropyl vinyl ether, n-butylvinyl ether, isobutyl vinyl ether, or tert-butyl vinyl ether, or apolymer derived from the polymer by the alkoxylation of its end;

(2) a nonionic polyamino acid having, as a constituent, any one monomerunit selected from amino acids such as glycine, alanine, valine,leucine, isoleucine, proline, phenylalanine, serine, threonine,asparagine, and glutamine, or a polymer derived from the polymer by thealkoxylation of its end;

(3) a nonionic synthetic polypeptide having, as a constituent, two ormore monomer units selected from amino acids such as glycine, alanine,valine, leucine, isoleucine, proline, phenylalanine, serine, threonine,asparagine, and glutamine, or a polymer derived from the polymer by thealkoxylation of its end;

(4) a nonionic polyester having, as a constituent, a monomer unitselected from glycolic acid and lactic acid, or a polymer derived fromthe polymer by the alkoxylation of its end;

(5) a nonionic polyether having, as a constituent, a monomer unitselected from glycols such as methylene glycol, ethylene glycol,n-propylene glycol, isopropylene glycol, and hydroxypropylene glycol, ora polymer derived from the polymer by the alkoxylation of its end;

(6) a nonionic natural polymer including sugars such as dextran, pectin,and pullulan, or a polymer derived from the polymer by the alkoxylationof its end;

(7) a nonionic modified natural polymer including celluloses such asmethylcellulose and hydroxypropylcellulose, or a polymer derived fromthe polymer by the alkoxylation of its end; and

(8) a block polymer or a graft copolymer having two or more differentpolymers selected from the polymers (1) to (7) as constituent units, ora copolymer derived from the copolymer by the alkoxylation of its end.

Of these nonionic water-soluble polymers, a nonionic polyether, anonionic polyester, a nonionic polyamino acid, or a nonionic syntheticpolypeptide, or a polymer derived from any of these polymers by thealkoxylation of the end is preferred. A nonionic polyether or a nonionicpolyester, or a polymer derived from any of these polymers by thealkoxylation of the end is more preferred. A nonionic polyether or anonionic monoalkoxy polyether is further preferred. Polyethylene glycolor monomethoxypolyethylene glycol is particularly preferred.Monomethoxypolyethylene glycol is most preferred.

The average molecular weight of the nonionic water-soluble polymer isnot particularly limited and is preferably 1000 to 12000, morepreferably 1000 to 5000, further preferably 1800 to 2200.

For example, any of the lipids listed in the paragraphs “2-1.Amphipathic lipid” and “2-2. Sterol” can be used in the lipid moiety.

Specific examples of the lipid bound with the nonionic water-solublepolymer can include, but are not limited to, diacylglycerol-boundmonomethoxypolyethylene glycol, phosphatidyl ethanolamine-boundmonomethoxypolyethylene glycol, and ceramide-boundmonomethoxypolyethylene glycol (U.S. Pat. No. 5,885,613).

More specific examples thereof can include

1,2-dilauroyl-sn-glycerol methoxypolyethylene glycol represented by thefollowing formula:

1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol represented bythe following formula:

1,2-dipalmitoyl-sn-glycerol methoxypolyethylene glycol represented bythe following formula:

1,2-distearoyl-sn-glycerol methoxypolyethylene glycol represented by thefollowing formula:

N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA; J.Controlled Release (2006) 112, p. 280-290) represented by the followingformula:

N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dipalmityloxypropyl-3-amine (PEG-C-DPA; J.Controlled Release (2006) 112, p. 280-290) represented by the followingformula:

N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearyloxypropyl-3-amine (PEG-C-DSA; J.Controlled Release (2006) 112, p. 280-290) represented by the followingformula:

mPEG2000-1,2-di-O-myristyl-sn3-carbomoylglyceride (PEG-DMG; described inExample 21 of WO2009/132131) represented by the following formula:

mPEG2000-1,2-di-O-palmityl-sn3-carbomoylglyceride (PEG-DPG; described inExample 21 of WO2009/132131) represented by the following formula:

and

mPEG2000-1,2-di-O-stearyl-sn3-carbomoylglyceride (PEG-DSG; described inExample 21 of WO2009/132131) represented by the following formula:

all of which have PEG having a molecular weight of approximately 2000.

Preferred examples thereof can include N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA) and1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol. More preferredexamples thereof can include N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA).

CH₃O(CH₂CH₂O)_(n′)CH₂CH₂O— in the structural formulas described aboverepresents the nonionic water-soluble polymer. Its average molecularweight is not particularly limited and is preferably 1000 to 12000, morepreferably 1000 to 5000, further preferably 1800 to 2200. n′ representsa numeric value estimated from the average molecular weight of thenonionic water-soluble polymer, and the number is not particularlylimited and is preferably 20 to 280, more preferably 20 to 120, furtherpreferably 35 to 50.

Normal PEG can also be used instead of or at the same time with thePEG-lipid described above as long as the PEG can prevent the aggregationof lipid particles. The PEG may be removed by dialysis beforeadministration if the lipid particle is stable after its production.

2-4. Other Constituents of the Lipid Particle

The lipid particle of the present invention can contain an additionalsubstance as long as the lipid particle maintains its structure. Oneexample of such a lipid particle can include a lipid particle containingone or two or more substances selected from a polyamide oligomer (seeU.S. Pat. No. 6,320,017), a peptide, a protein, and a detergent.

A constituent in the lipid particle of the present invention may bebound with a ligand having directivity to a target molecule.

Examples of the ligand can include: (1) a hormone, a growth factor, asuitable oligopeptide fragment thereof, or a low-molecular compound,which is bound with a particular cell receptor dominantly expressed by acell desired to be delivered; and (2) a polyclonal or monoclonalantibody, or a suitable fragment thereof (e.g., Fab or F(ab′)2), whichspecifically binds to an antigenic epitope dominantly found on a targetcell.

2-5. Compositional Ratio of Lipid Particle

The cationic lipid in the lipid particle according to the presentinvention is contained at approximately 20% to approximately 80%,preferably approximately 20% to approximately 70%, more preferablyapproximately 45% to approximately 65%, in terms of molar quantity withrespect to all lipids present in the lipid particle. As for the molarquantity of the cationic lipid used in the present invention, the lowerlimit is preferably 20%, more preferably 45%, with respect to all lipidspresent in the lipid particle, and the upper limit is preferably 80%,more preferably 70%, further preferably 65%, with respect to all lipidspresent in the lipid particle. The amphipathic lipid is contained atapproximately 0% to approximately 35%, preferably approximately 0% toapproximately 20%, more preferably approximately 5% to approximately15%, in terms of molar quantity with respect to all lipids present inthe lipid particle. As for the molar quantity of the amphipathic lipidused in the present invention, the lower limit is preferably 5% withrespect to all lipids present in the lipid particle, and the upper limitis preferably 35%, more preferably 20%, further preferably 15%, withrespect to all lipids present in the lipid particle. The sterol iscontained at approximately 0% to approximately 70%, preferablyapproximately 15% to approximately 70%, more preferably approximately15% to approximately 35%, in terms of molar quantity with respect to alllipids present in the lipid particle. As for the molar quantity of thesterol used in the present invention, the lower limit is preferably 15%with respect to all lipids present in the lipid particle, and the upperlimit is preferably 70%, more preferably 35%, with respect to all lipidspresent in the lipid particle. The lipid reducing aggregation duringlipid particle formation is contained at approximately 0.5% toapproximately 10%, preferably approximately 1% to approximately 10%,more preferably approximately 1.5% to approximately 10%, particularlypreferably approximately 1.5% to approximately 3%, in terms of molarquantity with respect to all lipids present in the lipid particle. Asfor the molar quantity of the lipid reducing aggregation during lipidparticle formation of the present invention, the lower limit ispreferably 0.5%, more preferably 1%, further preferably 1.1%, stillfurther preferably 1.2%, still further preferably 1.3%, particularlypreferably 1.4%, most preferably 1.5%, with respect to all lipidspresent in the lipid particle, and the upper limit is preferably 10%,more preferably 5%, further preferably 3%, with respect to all lipidspresent in the lipid particle.

When the amphipathic lipid, the sterol, the cationic lipid, and thelipid reducing aggregation during lipid particle formation are used inthe lipid particle of the present invention, the lipid composition ispreferably 20% or less of the amphipathic lipid, 15% to 70% of thesterol, 20% to 70% of the cationic lipid, and 1% to 10% of the lipidreducing aggregation during lipid particle formation, more preferably 5%to 15% or less of the amphipathic lipid, 15% to 40% or more of thesterol, 45% to 65% of the cationic lipid, and 1.5% to 3% of the lipidreducing aggregation during lipid particle formation, in terms of molarquantity.

Preferred examples of the lipid particle of the present invention caninclude a lipid particle having any ratio selected from molar ratios ofamphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation of 10:48:40:2, 10:38:50:2, 10:33:55:2,10:28:60:2, 15:33:50:2, 10:48.5:40:1.5, and 10:47.5:40:2.5. Morepreferred examples thereof can include a lipid particle having any ratioselected from molar ratios of amphipathic lipid:sterol:cationiclipid:lipid reducing aggregation during lipid particle formation of10:38:50:2, 10:33:55:2, 10:28:60:2, and 15:33:50:2.

3. Nucleic Acid Lipid Particle

The present invention provides a nucleic acid lipid particle comprisingthe lipid particle described in the preceding paragraph “2. Lipidparticle” and further a nucleic acid. The term “nucleic acid lipidparticle” means a complex of the lipid particle and a nucleic acid. Oneexample of the nucleic acid lipid particle in which the lipid particleis complexed with the nucleic acid can include a nucleic acid lipidparticle having a structure where a nucleic acid is buried in thebilayer of a lipid. One example of the nucleic acid lipid particle ofthe present invention can include a composition comprising the nucleicacid, the cationic lipid, the amphipathic lipid, the sterol, and thelipid reducing aggregation during lipid particle formation.

The ratio (N/P) of the number of molecules of the cationic lipid (N) tothe number of phosphorus atoms derived from the nucleic acid (P) in thenucleic acid lipid particle of the present invention is preferablyapproximately 2.0 to 15.0, more preferably approximately 2.0 to 12.0,further preferably 2.0 to 9.0, still further preferably 3.0 to 9.0. Thelower limit of the N/P ratio is preferably 2.0, more preferably 2.5,further preferably 3.0, and the upper limit thereof is preferably 15.0,more preferably 12.0, further preferably 9.0.

The nucleic acid lipid particle of the present invention has an averageparticle size of preferably approximately 30 nm to approximately 300 nm,more preferably approximately 30 nm to approximately 200 nm, furtherpreferably approximately 30 nm to approximately 100 nm. The averageparticle size refers to a volume-average particle size measured on thebasis of the principle of a dynamic light scattering method or the likeusing an apparatus such as Zeta Potential/Particle Sizer NICOMP™ 380ZLS(Particle Sizing Systems, LLC).

A nucleic acid that is degraded by nuclease under usual conditions isresistant to degradation by nuclease in an aqueous solution, whenpresent in the nucleic acid lipid particle of the present invention.

The nucleic acid lipid particle and a preparation method thereof aredisclosed in U.S. Pat. Nos. 5,753,613, 5,785,992, 5,705,385, 5,976,567,5,981,501, 6,110,745, and 6,320,017, and International Publication Nos.WO 96/40964 and WO 07/012191.

In the present specification, the term “nucleic acid”,“oligonucleotide”, or “polynucleotide” refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in any form of asingle strand, a double strand, and a triple strand.

A specific nucleic acid sequence also encompasses, implicitly,conservatively modified variants (e.g., degenerate codon substitutes),alleles, orthologs, SNPs, and complementary sequences thereof, andexplicitly specified sequences, unless otherwise specified.

DNA may be in the form of an antisense, a plasmid DNA, a portion of theplasmid DNA, a DNA concentrated beforehand, a polymerase chain reaction(PCR) product, a vector (P1, PAC, BAC, YAC, or artificial chromosome),an expression cassette, a chimeric sequence, a chromosomal DNA, or aderivative of these groups.

In the present specification, the term “nucleic acid” is used for all ofa gene, a plasmid, a cDNA, a messenger RNA (mRNA), and an interferenceRNA molecule (e.g., synthetic siRNA or siRNA expressed from a plasmid).

3-1. Nucleic Acid that Forms Nucleic Acid Lipid Particle

The nucleic acid that forms the nucleic acid lipid particle of thepresent invention can include any form known to those skilled in theart. Specific examples of such a form of the nucleic acid can include asingle-stranded DNA, a single-stranded RNA, and a single-strandedpolynucleotide of a DNA and an RNA mixed with each other. Specificexamples of other forms of the nucleic acid can include adouble-stranded polynucleotide consisting of a double-stranded DNA, adouble-stranded RNA, a DNA-RNA hybrid polynucleotide, or twopolynucleotides of a DNA and an RNA mixed with each other.

3-2. Nucleoside or Nucleotide

Each nucleoside or nucleotide constituting the nucleic acid contained inthe nucleic acid lipid particle of the present invention includesnatural one as well as a modified nucleoside or a modified nucleotideprepared by chemical modification. Examples of the modified nucleosideor nucleotide include a sugar-modified nucleoside or nucleotide, anucleobase-modified nucleoside or nucleotide, a backbone-modifiednucleoside or nucleotide, and combinations thereof (see e.g., NucleicAcid Research, 1997, Vol. 25, No. 22, 4429-4443).

In the present specification, the “natural nucleoside” refers to a2′-deoxynucleoside such as 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, 2′-deoxy-5-methylcytidine, and thymidine or aribonucleoside such as adenosine, guanosine, cytidine, 5-methylcytidine,and uridine. Moreover, the “oligonucleotide” refers to anoligonucleotide composed of a compound in which the sugar moiety of thenucleoside forms an ester with phosphoric acid. In the presentspecification, the terms “oligonucleotide” and “polynucleotide” are usedinterchangeably.

In the present specification, 2′-deoxyadenosine may be referred to asA^(t); 2′-deoxyguanosine may be referred to as G^(t); 2′-deoxycytidinemay be referred to as C^(t); 2′-deoxy-5-methylcytidine may be referredto as 5meC^(t); thymidine may be referred to as T^(t); 2′-deoxyuridinemay be referred to as U^(t); adenosine may be referred to as A^(rt);guanosine may be referred to as G^(rt); cytidine may be referred to asC^(rt); 5-methylcytidine may be referred to as 5meC^(rt); and uridinemay be referred to as U^(rt). Moreover, in the present specification, a2′-deoxyadenosine nucleotide may be referred to as A^(p); a2′-deoxyguanosine nucleotide may be referred to as G^(p); a2′-deoxycytidine nucleotide may be referred to as C^(p); a2′-deoxy-5-methylcytidine nucleotide may be referred to as 5meC^(p); athymidine nucleotide may be referred to as T^(p); a 2′-deoxyuridinenucleotide may be referred to as U^(p); an adenosine nucleotide may bereferred to as A^(rp); a guanosine nucleotide may be referred to asG^(rp); a cytidine nucleotide may be referred to as C^(rp); a5-methylcytidine nucleotide may be referred to as 5meC^(rp); and auracil nucleotide may be referred to as U^(rp).

In the present specification, where there are phosphorothioate esterforms instead of phosphoester forms of a nucleotide, a counterpart ofA^(p) may be referred to as A^(s); a counterpart of G^(p) may bereferred to as G^(s); a counterpart of C^(p) may be referred to asC^(s); a counterpart of 5meC^(p) may be referred to as 5meC^(s); acounterpart of T^(p) may be referred to as T^(s); a counterpart of U^(p)may be referred to as U^(s); a counterpart of A^(rp) may be referred toas A^(rs); a counterpart of G^(rp) may be referred to as G^(rs); acounterpart of C^(rp) may be referred to as C^(rs); a counterpart of5meC^(rp) may be referred to as 5meC^(rs); and a counterpart of U^(rp)may be referred to as U^(rs).

In the present specification, the term “sugar-modified nucleoside”refers to a nucleoside modified at its sugar moiety. Examples of thesugar-modified nucleoside include 2′-O-methyl nucleoside,2′-O,4′-C-ethylene nucleoside (ENA), and 2′-O,4′-C-methylene nucleotide(BNA/LNA).

In particular, examples of 2′-O-methyl modification include 2′-O-methylnucleoside and 2′-O-methyl nucleotide: a counterpart of A^(rt) may bereferred to as A^(mlt); a counterpart of G^(rt) may be referred to asG^(mlt); a counterpart of C^(rt) may be referred to as C^(mlt); acounterpart of 5meC^(rt) may be referred to as 5meC^(mlt); a counterpartof U^(rt) may be referred to as U^(mlt); a counterpart of A^(rp) may bereferred to as A^(mlp); a counterpart of G^(rp) may be referred to asG^(mlp); a counterpart of C^(rp) may be referred to as C^(mlp); acounterpart of 5meC^(rp) may be referred to as 5meC^(mlp); a counterpartof U^(rp) may be referred to as U^(mlp); a counterpart of A^(rs) may bereferred to as A^(mls); a counterpart of G^(rs) may be referred to asG^(mls); a counterpart of C^(rs) may be referred to as C^(mls); acounterpart of 5meC^(s) may be referred to as 5meC^(mls); and acounterpart of U^(rs) may be referred to as U^(mls).

In the Sequence Listing attached to the present specification, “cm” inthe item <223> of each sequence represents 2′-O-methylcytidine; “um”represents 2′-O-methyluridine; and “gm” represents 2′-O-methylguanosine.

In the present specification, the 2′-O,4′-C-ethylene nucleotide unit andthe “ENA unit” refer to those nucleosides and nucleotides having an ENAand also refer to nucleosides and nucleotides having an ENA unit: acounterpart of A^(t) may be referred to as A^(2t); a counterpart ofA^(p) may be referred to as A^(e2p); a counterpart of A^(s) may bereferred to as A^(e2s); a counterpart of G^(t) may be referred to asG^(2t); a counterpart of G^(p) may be referred to as G^(e2p); acounterpart of G^(s) may be referred to as G^(e2s); a counterpart of5meC^(t) may be referred to as C^(2t); a counterpart of 5meC^(p) may bereferred to as C^(e2p); a counterpart of 5meC^(s) may be referred to asC^(e2s); a counterpart of T^(t) may be referred to as T^(2t); acounterpart of T^(p) may be referred to as T^(e2p); and a counterpart ofT^(s) may be referred to as T^(e2s).

In the present specification, the 2′-0,4′-C-methylene nucleotide unitand the “2′,4′-BNA/LNA unit” refer to those nucleosides and nucleotideshaving a 2′,4′-BNA/LNA and also refer to nucleosides and nucleotideshaving a 2′,4′-BNA/LNA unit: a counterpart of A^(t) may be referred toas A^(1t); a counterpart of A^(p) may be referred to as A^(e1p); acounterpart of A^(s) may be referred to as A^(e1s;) a counterpart ofG^(t) may be referred to as G^(1t); a counterpart of G^(p) may bereferred to as G^(e1p); a counterpart of G^(s) may be referred to asG^(e1s); a counterpart of 5meC^(t) may be referred to as C^(1t); acounterpart of 5meC^(p) may be referred to as C^(e1p); a counterpart of5meC^(s) may be referred to as C^(e1s); a counterpart of T^(t) may bereferred to as T^(1t); a counterpart of T^(p) may be referred to asT^(e1p); and a counterpart of T^(s) may be referred to as T^(e1s).

Hereinafter, the structural formula of each nucleotide is shown.

3-3. Target Gene

In the present specification, the “target gene” is not particularlylimited as long as it is RNA in cells, tissues, or individuals to whichor to whom this gene is introduced (hereinafter, they may be referred toas “recipients”). The target gene may be mRNA that is translated into aprotein or may be non-coding RNA that is not translated into a protein.Examples of the non-coding RNA include functional RNA, for example, anuntranslated region of mRNA, tRNA, rRNA, mRNA-like non-coding RNA(mRNA-like ncRNA), long non-coding RNA (long ncRNA), small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), and microRNA (miRNA).Specifically, the target gene may be endogenous to the recipients forintroduction or may be exogenous and introduced thereto by an approachsuch as gene transfer. It may also be a gene present on a chromosome oron an extrachromosomal gene. Examples of the exogenous gene include, butare not limited to, those derived from viruses, bacteria, fungi, andprotozoans, which can infect the recipients. The function of the genemay be known or unknown.

Examples of such a target gene can include genes whose expression isspecifically increased and/or which are specifically mutated in patientshaving a particular disease. Examples of the disease can include centralnervous system disease (e.g., Alzheimer's disease, dementia, and eatingdisorders), inflammatory disease (e.g., allergy, rheumatism,osteoarthritis, and lupus erythematosus), cardiovascular disease (e.g.,hypertension, cardiomegaly, angina pectoris, arteriosclerosis, andhypercholesterolemia), cancer (e.g., non-small cell lung cancer, ovariancancer, prostatic cancer, gastric cancer, bladder cancer, breast cancer,uterine cervix cancer, colon cancer, rectal cancer, liver cancer, kidneycancer, pancreatic cancer, and malignant melanoma), respiratory disease(e.g., pneumonia, bronchitis, asthma, chronic obstructive pulmonarydisease, and lung fibrosis), diabetes mellitus, diabetic retinopathy,diabetic nephropathy, anemia (e.g., anemia associated with chronicdisease and iron-refractory iron deficiency anemia), age-related maculardegeneration, immunological disease (e.g., Crohn's disease, atopicdermatitis, autoimmune disease, immunodeficiency, and leukemia),liver/gallbladder disease (e.g., non-alcoholic steatohepatitis, livercirrhosis, hepatitis, liver failure, cholestasis, and calculus),gastrointestinal disease (e.g., ulcer, enteritis, and malabsorption),infection, adiposity, and fibrosis (e.g., lung fibrosis, liver fibrosis,renal fibrosis, and myelofibrosis). Examples of causative genes of thesediseases can include, but are not limited to, kinesin spindle protein(KSP), vascular endothelial growth factor (VEGF), transthyretin (TTR),proprotein convertase subtilisin/kexin type 9 (PCSK9), polo-like kinase1 (PLK-1), ApoB-100, ribonucleotide reductase M2 subunit (RRM2),clusterin, heat shock protein 27 (Hsp27), survivin, eukaryoticinitiation factor-4E (eIF-4E), intercellular adhesion molecule 1(ICAM-1), the alpha subunit of the interleukin 4 receptor (IL-4R-alpha),Factor XI, Factor VII, N-ras, H-ras, K-ras, bcl-2, bcl-xL, Her-1, Her-2,Her-3, Her-4, MDR-1, human β-catenin gene, DDX3 (DEAD (Asp-Glu-Ala-Asp)box polypeptide 3, X-linked), Myeloid Cell Leukemia Sequence 1 (MCL1)gene, PKR (Eif2ak2), Hsp47 (Serpinh1), Hepcidin, active protein c (APC),signal transducer and activator of transcription (STAT3), and Collagen,type I, alpha 1 (Col1A1).

3-4. Double-stranded Polynucleotide

When the nucleic acid contained in the nucleic acid lipid particle ofthe present invention is a nucleic acid having an RNA interferenceeffect on a target gene, the nucleic acid is not limited by itsstructure and chemical modification as long as it has an RNAinterference effect. Examples thereof can include siRNA (see e.g.,WO2002/044321 and Current Opinion in Chemical Biology 570-579), AtuRNAiconsisting of a polynucleotide containing alternately bound RNAs and2′-OMeRNAs (see e.g., WO2004/015107), a double-stranded polynucleotidein which sense and antisense strands of polynucleotides containingalternately bound DNAs and 2′-OMeRNAs form a double strand byWatson-Crick base pairing between different types of nucleic acids (seee.g., WO2010/001909), which is described below in the paragraph 3-4-1, anucleic acid consisting of a terminally modified polynucleotide (seee.g., WO2010/052715), which is described below in the paragraph 3-4-2,and a single-stranded polynucleotide in which the 5′-end of an antisensestrand polynucleotide and the 3′-end of a sense strand polynucleotideare bound to each other via a linker to form a single strand, whichfurther intramolecularly forms a double-stranded structure byWatson-Crick base pairing (see e.g., WO2012/074038), which is describedbelow in the paragraph 3-4-3.

The structures of these polynucleotides are shown in FIG. 4.

In the present specification, the phrase “having a nucleotide sequenceidentical to a target gene” refers to having a sequence identical to atleast a partial nucleotide sequence of the target gene. It includes acompletely identical sequence and also includes a substantiallyidentical sequence as long as the resulting polynucleotide has an RNAinterference effect and/or a gene expression inhibitory effect on thetarget gene. The phrase “having a nucleotide sequence complementary tothe target gene” refers to having a sequence complementary to at least apartial nucleotide sequence of the target gene. It includes a completelycomplementary sequence and also includes a substantially identicalsequence as long as the resulting polynucleotide has an RNA interferenceeffect and/or a gene expression inhibitory effect on the target gene.When the target gene is known to have SNPs or the like, a sequencehaving these variations is also included as an identical nucleotidesequence. In the present specification, a polynucleotide that comprisesa nucleotide sequence complementary to a target gene and has an RNAinterference effect and/or a gene expression inhibitory effect on thetarget gene is referred to as a polynucleotide against the target gene.

The nucleotide sequence of the nucleic acid contained in the nucleicacid particle of the present invention is not particularly limited aslong as it has an RNA interference effect and/or a gene expressioninhibitory effect on the target gene. For example, the nucleotidesequence can be determined by determining the sequences of sense andantisense strands on the basis of a sequence predicted to have RNAinterference effect on the target gene using computer software (e.g.,GENETYX(R): manufactured by GENETYX CORPORATION), and can also bedetermined by further confirming the RNA interference effect and/or geneexpression inhibitory effect of a polynucleotide prepared on the basisof the selected sequence.

The respective chain lengths of the sense and antisense strands of thedouble-stranded polynucleotide having an RNA interference effect may beany length from 10 nucleotides to the full length of the open readingframe (ORF) of the target gene as long as the resulting polynucleotidehas an RNA interference effect and/or a gene expression inhibitoryeffect. The respective chain lengths of the sense and antisense strandsare preferably any length from 18 nucleotides to the full length of theopen reading frame (ORF) of the target gene, more preferably 10 to 100nucleotides, further preferably 15 to 30 nucleotides.

In the case of using a double-stranded polynucleotide in which sense andantisense strands of polynucleotides containing alternately bound DNAsand 2′-OMeRNAs are bound by Watson-Crick base pairing between differenttypes of nucleic acids (WO2010/001909) as the double-strandedpolynucleotide having an RNA interference effect, the chain length ofthe sense strand is preferably 18 to 21 nucleotides, more preferably 18or 19 nucleotides. The chain length of the antisense strand ispreferably 19 to 21 nucleotides, more preferably 21 nucleotides. Thispolynucleotide does not have to be a double-stranded structure as awhole and also includes those partially overhanging at the 5′- and/or3′-ends. The overhanging end has 1 to 5 nucleotides, preferably 1 to 3nucleotides, more preferably 2 nucleotides. Most preferred examples ofthe polynucleotide include a polynucleotide having a structure where the3′-end of the antisense strand polynucleotide overhangs by 2 nucleotides(overhang structure), and having 18 base pairs.

3-4-1. Polynucleotide Containing Alternately Bound DNAs and 2′-OMeRNAs

One example of the nucleic acid contained in the nucleic acid lipidparticle of the present invention can include a double-strandedpolynucleotide in which sense and antisense strands of polynucleotidescontaining alternately bound DNAs and 2′-OMeRNAs are bound byWatson-Crick base pairing between different types of nucleic acids.

Specific examples of such a double-stranded polynucleotide include

a double-stranded polynucleotide constituted by

a sense strand CT-169 described in Example 51 of WO2010/001909:

HO-G^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-A^(mlp)-G^(p)-A^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlt)-H(SEQ ID NO: 1 of the Sequence Listing) and

an antisense strand CT-157 described in Example 45 thereof:

HO—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 2 of the Sequence Listing); and

a double-stranded polynucleotide constituted by

a sense strand CT-103 described in Example 20 of WO2010/001909:

HO-G^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-A^(mlp)-G^(p)-A^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlp)-A^(t)-H(SEQ ID NO: 3 of the Sequence Listing) and

an antisense strand CT-157 described in Example 45 thereof:

HO—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 2 of the Sequence Listing).

3-4-2. Modified Double-stranded Polynucleotide

In the case of using a nucleic acid having an RNA interference effect asthe nucleic acid contained in the nucleic acid lipid particle, anotherexample thereof can include a nucleic acid modified at the end of itspolynucleotide as long as the nucleic acid has the RNA interferenceeffect. Examples thereof can include a double-stranded polynucleotidederived from a double-stranded polynucleotide having an RNA interferenceeffect (e.g., siRNA, AtuRNAi, or a double-stranded polynucleotide inwhich sense and antisense strands of polynucleotides containingalternately bound DNAs and 2′-OMeRNAs are bound by Watson-Crick basepairing between different types of nucleic acids (see e.g.,WO2010/001909)), and 5′-modified with aryl phosphate at the phosphategroup of the 5′-end of its antisense strand (see e.g., WO2010/052715).

Specific examples of such a modified double-stranded polynucleotideinclude

a modified double-stranded polynucleotide constituted by

a sense strand CT-169 described in Example 1 of WO2010/052715:

HO-G^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-A^(mlp)-G^(p)-A^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlt)-H(SEQ ID NO: 1 of the Sequence Listing) and

an antisense strand having any one sequence selected from the following(1) to (3):

(1) an antisense strand CT-292 described in Example 17 thereof:

X—P(═O)(OH)—O-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 4 of the Sequence Listing) wherein

X is represented by the following formula:

(2) an antisense strand CT-315 described in Example 26 thereof:X—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 5 of the Sequence Listing) wherein

X is represented by the following formula:

and

(3) an antisense strand CT-387 described in Example 83 thereof:

X—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 6 of the Sequence Listing) wherein

X is represented by the following formula:

3-4-3. Modified Single-stranded Polynucleotide

The nucleic acid contained in the nucleic acid lipid particle alsoincludes a polynucleotide that has a sense strand polynucleotide againstthe target gene and an antisense strand polynucleotide having anucleotide sequence complementary to the sense strand polynucleotide andhas a single-stranded structure where the 5′-end of the antisense strandpolynucleotide and the 3′-end of the sense strand polynucleotide arebound to each other through a phosphodiester structure formed via alinker as long as the polynucleotide has an RNA interference effect (seee.g., WO2012/074038).

Specific examples of such a compound can include:

a polynucleotide CT-454 described in Example 12 of WO2012/074038:

HO-G^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-A^(mlp)-G^(p)-A^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlp)-X—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 7 (sense strand region) and SEQ ID NO: 8 (antisense strandregion) of the Sequence Listing);a polynucleotide HS-005 described in Example 28 thereof:HO—C^(p)-G^(mlp)-A^(p)-G^(mlp)-A^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-A^(mlp)-X—P(═O)(OH)—O-U^(mlp)-T^(p)-A^(mlp)-G^(p)-C^(mlp)-A^(p)-C^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-U^(mlp)-C^(p)-U^(mlp)-C^(p)-G^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 9 (sense strand region) and SEQ ID NO: 10 (antisense strandregion) of the Sequence Listing);a polynucleotide HS-006 described in Example 29 thereof:HO—C^(p)-A^(mlp)-G^(p)-A^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlp)-T^(p)-G^(mlp)-G^(p)-G^(mlp)-T^(p)-G^(mlp)-C^(p)-U^(mlp)-A^(p)-U^(mlp)-X—P(═O)(OH)—O-U^(mlp)-A^(p)-U^(mlp)-A^(p)-G^(mlp)-C^(p)-A^(mlp)-C^(p)-C^(mlp)-C^(p)-A^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-T^(p)-C^(mlp)-T^(p)-G^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 11 (sense strand region) and SEQ ID NO: 12 (antisense strandregion) of the Sequence Listing);a polynucleotide HS-005s described in Example 30 thereof:HO—C^(p)-G^(mlp)-A^(p)-G^(mlp)-A^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-A^(mlp)-X—P(═O)(OH)—O-U^(mlp)-T^(p)-A^(mlp)-G^(p)-C^(mlp)-A^(p)-C^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-U^(mlp)-C^(p)-U^(mlp)-C^(p)-G^(mlp)-T^(ps)-U^(mlt)-H(SEQ ID NO: 13 (sense strand region) and SEQ ID NO: 14 (antisense strandregion) of the Sequence Listing); anda polynucleotide HS-006s described in Example 31 thereof:HO—C^(p)-A^(mlp)-G^(p)-A^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlp)-T^(p)-G^(mlp)-G^(p)-G^(mlp)-T^(p)-G^(mlp)-C^(p)-U^(mlp)-A^(p)-U^(mlp)-X—P(═O)(OH)—O-U^(mlp)-A^(p)-U^(mlp)-A^(p)-G^(mlp)-C^(p)-A^(mlp)-C^(p)-C^(mlp)-C^(p)-A^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-T^(p)-C^(mlp)-T^(p)-G^(mlp)-T^(ps)-U^(mlt)-H(SEQ ID NO: 15 (sense strand region) and SEQ ID NO: 16 (antisense strandregion) of the Sequence Listing), whereinX is represented by the following formula:

The terminal methylene group of X binds to the 3′-end of the sensestrand polynucleotide to form a phosphodiester bond, and the oxygen atombonded to the phenyl group binds to the 5′-end of the antisense strandpolynucleotide to form a phosphodiester bond.

3-4-3. Single-stranded RNA

The nucleic acid contained in the nucleic acid lipid particle can be anysingle-stranded RNA without particular limitations and also includesmRNA that is translated into a protein. In order to improve translationefficiency, its sequence can also contain a cap structure (e.g.,m7GpppG) or an internal ribosome entry site (IRES) at the 5′-end and/ora poly-A tail at the 3′-end. Further, the 3′ and/or 5′ untranslatedregion can contain a sequence that contributes to the stabilization of aprotein, or a sequence that promotes translation.

The single-stranded RNA can be produced by in vitro transcriptionreaction from a DNA having a desired nucleotide sequence. Enzymes,buffer solutions, and a nucleoside-5′-triphosphate mixture(adenosine-5′-triphosphate (ATP), guanosine-5′-triphosphate (GTP),cytidine-5′-triphosphate (CTP), and uridine-5′-triphosphate (UTP))necessary for the in vitro transcription are commercially available(AmpliScribe T7 High Yield Transcription Kit (Epicentre), mMESSAGEmMACHINE T7 Ultra Kit (Life Technologies, Inc.), etc.). The DNA used forproducing the single-stranded RNA is a cloned DNA, and, for example, aplasmid DNA or a DNA fragment is used.

In order to improve stability and further obtain an mRNA having reducedimmunogenicity, a modified nucleotide may be introduced into the mRNA byusing a modified nucleoside-5′-triphosphate together with an unmodifiednucleoside-5′-triphosphate in the in vitro transcription reaction(Kormann, M. (2011) Nature Biotechnology 29, 154-157.). Examples of themodified uridine-5′-triphosphate used can include2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,4′-thiouridine-5′-triphosphate, and pseudouridine-5′-triphosphate.Examples of the modified cytidine-5′-triphosphate used can include5-methylcytidine-5′-triphosphate and 4-thiocytidine-5′-triphosphate. Themodified uridine-5′-triphosphate and the modifiedcytidine-5′-triphosphate may be used at the same time as the modifiednucleoside-5′-triphosphates.

The ratio between the unmodified uridine-5′-triphosphate and themodified uridine-5′-triphosphate is preferably 50 to 95% of theunmodified uridine-5′-triphosphate and 5 to 50% of the modifieduridine-5′-triphosphate, more preferably 70 to 95% of the unmodifieduridine-5′-triphosphate and 5 to 30% of the modifieduridine-5′-triphosphate. The ratio between the unmodifiedcytidine-5′-triphosphate and the modified cytidine-5′-triphosphate ispreferably 50 to 95% of the unmodified cytidine-5′-triphosphate and 5 to50% of the modified cytidine-5′-triphosphate, more preferably 70 to 95%of the unmodified cytidine-5′-triphosphate and 5 to 30% of the modifiedcytidine-5′-triphosphate.

The single-stranded RNA containing the modified nucleotide (or modifiednucleoside) obtained by the in vitro transcription reaction using themodified nucleoside-5′-triphosphate can be completely hydrolyzed withnuclease (if necessary, which can also be dephosphorylated withphosphatase) and analyzed using, for example, thin-layer chromatography(TLC) or high-performance liquid chromatography (HPLC) to determine thecontents of the modified nucleotide and an unmodified nucleotide (or thecontents of the modified nucleoside and an unmodified nucleoside).

The ratio between the unmodified uridine and the modified uridine ispreferably 50 to 95% of the unmodified uridine and 5 to 50% of themodified uridine, more preferably 70 to 95% of the unmodified uridineand 5 to 30% of the modified uridine. The ratio between the unmodifiedcytidine and the modified cytidine is preferably 50 to 95% of theunmodified cytidine and 5 to 50% of the modified cytidine, morepreferably 70 to 95% of the unmodified cytidine and 5 to 30% of themodified cytidine.

The single-stranded RNA is used for treating a disease or supplying abeneficiary protein. The single-stranded RNA is delivered to an organresponsible for the disease through the nucleic acid lipid particle ofthe present invention and further transported into the cytoplasm. Whenthe single-stranded RNA encodes a protein, the single-stranded RNA istranslated into the protein in the cytoplasm so that this protein bringsabout the curing of the disease.

A target of the treatment of the disease may be the absence of a proteindue to gene mutation or a decreased supply of the protein. Even if theprotein is present, the mutation of its gene causes mutation in theprotein, and this variant protein may have functions weaker than thoseof the natural protein in some cases. For such deletion or deficiency ofthe protein, a single-stranded RNA encoding the protein can be used tobring about the curing of the disease. Examples of the disease that canbe cured using the single-stranded RNA can include a disease caused by agenetic defect (genetic disease), and a disease caused by the absence ofa protein in the body due to organ failure.

Examples of the disease caused by a genetic defect (genetic disease)(gene name is indicated within the parentheses) can include glycogenstorage disease type Ia (glucose-6-phosphatase), glycogen storagedisease type Ib (glucose-6-phosphate translocase), glycogen storagedisease type III (amylo-1,6-glucosidase), glycogen storage disease typeIV (amylo-1,4→1,6 transglucosylase), glycogen storage disease type VI(liver phosphorylase), glycogen storage disease type IX, glycogenstorage disease type VIII (liver phosphorylase kinase), α1-antitrypsindeficiency (α1-antitrypsin), congenital hemochromatosis, hepcidindeficiency (hepcidin), hemophilia A and B (coagulation factors VIII andIX, respectively), congenital anticoagulant deficiency (protein C,inactivator of coagulation factors Va and VIIIa), thromboticthrombocytopenic purpura (ADAMTS13), congenital amegakaryocyticthrombocytopenia, and thrombopoietin deficiency (thrombopoietin).Examples of the disease caused by the absence of a protein in the bodydue to organ failure can include erythropoietin (EPO) growth hormone(somatotropin or hGH).

3-5. Method for Producing Nucleic Acid Lipid Particle

The method for producing the nucleic acid lipid particle of the presentinvention is not particularly limited as long as the nucleic acid lipidparticle can be produced by the method. The nucleic acid lipid particlecan be produced, for example, by a method such as a thin film method, areverse-phase evaporation method, an ethanol injection method, an etherinjection method, a dehydration-rehydration method, a detergent dialysismethod, a hydration method, or a freezing-thawing method. Morespecifically, the nucleic acid lipid particle can be produced by theethanol injection method described below.

Hydrophobic materials such as the cationic lipid, the amphipathic lipid,and the lipid reducing aggregation during lipid particle formation aredissolved in 50 to 90% ethanol. On the other hand, hydrophilic materialssuch as the nucleic acid are dissolved in a buffer solution of pH 3 to6.

The solution of the lipids in ethanol and the aqueous solution of thenucleic acid are mixed at a volume ratio of 1:20 to 1:1 to form a lipidparticle and to form a nucleic acid lipid particle through theelectrostatic interaction between the negatively charged nucleic acidand the positively charged cationic lipid. As a result, a crudedispersion of the nucleic acid lipid particle is obtained.

In another aspect, the solution of the lipids in ethanol is mixed with abuffer solution free from the nucleic acid to form a lipid particle.Then, the lipid particle may be mixed with the aqueous solution of thenucleic acid to form a nucleic acid lipid particle.

Subsequently, ethanol and free nucleic acids contained in the obtainedcrude dispersion of the nucleic acid lipid particle are removed by amethod such as ultrafiltration or dialysis to obtain a stable nucleicacid lipid particle.

Examples of such a nucleic acid lipid particle can include a nucleicacid lipid particle comprising the constituents at any molar ratioselected from the group consisting of the following (a) to (g):

(a) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:48:40:2,

(b) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:38:50:2,

(c) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:33:55:2,

(d) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:28:60:2,

(e) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=15:33:50:2,

(f) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:48.5:40:1.5, and

(g) amphipathic lipid:sterol:cationic lipid:lipid reducing aggregationduring lipid particle formation=10:47.5:40:2.5.

The ratio (N/P) of the number of molecules of the cationic lipid (N) tothe number of phosphorus atoms derived from the nucleic acid (P) in thenucleic acid lipid particle is preferably approximately 2.0 to 15.0,more preferably approximately 2.0 to 12.0, further preferably, 2.0 to9.0, still further preferably 3.0 to 9.0. The lower limit of the N/Pratio is preferably 2.0, more preferably 2.5, further preferably 3.0,and the upper limit thereof is preferably 15.0, more preferably 12.0,further preferably 9.0.

4. Pharmaceutical Composition Containing Nucleic Acid Lipid Particle

The nucleic acid lipid particle of the present invention can be used ina pharmaceutical product as long as the nucleic acid lipid particle hasan RNA interference effect and/or a gene inhibitory effect on a targetgene.

The pharmaceutical product is not particularly limited as long as thepharmaceutical product is for the treatment or prevention of a diseasederived from the expression of a target gene. Preferred examples thereofinclude pharmaceutical products for treating or preventing centralnervous system disease (e.g., Alzheimer's disease, dementia, and eatingdisorders), inflammatory disease (e.g., allergy, rheumatism,osteoarthritis, and lupus erythematosus), cardiovascular disease (e.g.,hypertension, cardiomegaly, angina pectoris, arteriosclerosis, andhypercholesterolemia), cancer (e.g., non-small cell lung cancer, ovariancancer, prostatic cancer, gastric cancer, bladder cancer, breast cancer,uterine cervix cancer, colon cancer, rectal cancer, liver cancer, kidneycancer, pancreatic cancer, and malignant melanoma), respiratory disease(e.g., pneumonia, bronchitis, asthma, chronic obstructive pulmonarydisease, and lung fibrosis), diabetes mellitus, diabetic retinopathy,diabetic nephropathy, anemia (e.g., anemia associated with chronicdisease, iron-refractory iron deficiency anemia, and anemia of cancer),age-related macular degeneration, immunological disease (e.g., Crohn'sdisease, atopic dermatitis, autoimmune disease, immunodeficiency, andleukemia), liver/gallbladder disease (e.g., non-alcoholicsteatohepatitis, liver cirrhosis, hepatitis, liver failure, cholestasis,and calculus), gastrointestinal disease (e.g., ulcer, enteritis, andmalabsorption), infection, adiposity, and fibrosis (e.g., lung fibrosis,liver fibrosis, renal fibrosis, and myelofibrosis). The pharmaceuticalproduct is more preferably for the treatment or prevention of cancer(e.g., non-small cell lung cancer, ovarian cancer, prostatic cancer,gastric cancer, bladder cancer, breast cancer, uterine cervix cancer,colon cancer, rectal cancer, liver cancer, kidney cancer, pancreaticcancer, and malignant melanoma), respiratory disease (e.g., pneumonia,bronchitis, asthma, chronic obstructive pulmonary disease, and lungfibrosis), and/or liver/gallbladder disease (e.g., non-alcoholicsteatohepatitis, liver cirrhosis, hepatitis, liver failure, cholestasis,and calculus). The pharmaceutical product is further preferably for thetreatment or prevention of cancer (colon cancer, rectal cancer, andliver cancer), anemia (e.g., anemia associated with chronic disease,iron-refractory iron deficiency anemia, and anemia of cancer), liverdisease (non-alcoholic steatohepatitis, liver cirrhosis, and hepatitis),gallbladder disease (cholestasis), and fibrosis (lung fibrosis, liverfibrosis, and renal fibrosis).

The nucleic acid lipid particle of the present invention can be used ina pharmaceutical product as long as the single-stranded RNA is used fortreating a disease or supplying a beneficiary protein. Examples of thiscase are shown in the paragraph 3-4-3.

The nucleic acid lipid particle of the present invention can beadministered either alone or in a mixture with a physiologicallyacceptable carrier selected according to an administration route and astandard pharmaceutical practice.

In general, standard saline is used as a pharmaceutically acceptablecarrier.

Other preferred carriers include, for example, water, buffered water,0.4% salt solutions, and 0.3% glycine and also include albumins,lipoproteins, and glycoproteins such as globulins in order to enhancestability.

The pharmaceutical carriers are generally added after particleformation. Thus, after the particle formation, the particle can bediluted in a pharmaceutically acceptable carrier such as standardsaline.

The particle in a pharmaceutical formulation can have a very wideconcentration range. Specifically, the concentration is less thanapproximately 0.05%, usually approximately 2 to 5%, or from at leastapproximately 2 to 5% to approximately 10 to 30%, of the weight, and isselected mainly from the volume, viscosity, or the like of a liquidaccording to a selected specific administration mode. For example, theconcentration may be elevated such that a load of the liquid associatedwith treatment may be decreased. This is particularly desirable forpatients with atherosclerosis-related congestive heart failure or severehypertension. Alternatively, a particle constituted by an irritatinglipid can be diluted to a low concentration, which can reduceinflammation at an administration site.

Typically, the concentration of the nucleic acid in the nucleic acidlipid particle is approximately 1 to 20%, more preferably approximately3 to 10%.

The pharmaceutical composition of the present invention may besterilized by a usual well-known sterilization technique. An aqueoussolution can be packaged for use or can be filtered and freeze-driedunder aseptic conditions. The freeze-dried preparation is combined withan aseptic aqueous solution before administration. The composition cancontain pharmaceutically acceptable auxiliaries necessary forapproaching a physiological state, for example, a pH adjustor and abuffering agent (e.g., sodium acetate, sodium lactate, sodium chloride,potassium chloride, and calcium chloride) and an osmotic regulator.

In addition, the particle suspension may contain a lipid-protectingagent that protects lipids from free radicals and lipid peroxidationdamage during storage. A lipophilic free radical quencher such as alphatocopherol and a water-soluble ion-specific chelating agent such asferrioxamine are preferred.

Other examples of the use of the nucleic acid lipid particle include,but are not limited to, gels, oils, and emulsions. The nucleic acidlipid particle can also be incorporated into a wide range of localdosage forms. For example, the suspension containing the nucleic acidlipid particle can be formulated and administered as a local cream,paste, ointment, gel, lotion, or the like.

The nucleic acid lipid particle of the present invention also provides amethod for transferring a nucleic acid (e.g., plasmid or siRNA) into acell. The method is carried out in vitro or in vivo by first forming theparticle as described above and then contacting the particle with a cellfor a time long enough to deliver the nucleic acid into the cell.

The nucleic acid lipid particle of the present invention can be adsorbedto almost every type of cell with which the nucleic acid lipid particleis mixed or contacted. Once the nucleic acid lipid particle is adsorbedthereto, the particle can achieve any of the following events: theparticle is endocytosed by the cell moiety; the cell membrane isreplaced with the lipid; and the particle is fused with the cell.

The delivery or uptake of the nucleic acid moiety of the particle takesplace through any one of these routes. Particularly, when the fusionoccurs, the particle membrane is incorporated into the cell membrane sothat the contents in the particle are combined with the intracellularfluid.

The nucleic acid lipid particle of the present invention is useful forthe treatment or prevention of every sign, disease, or symptom involvedin or responding to the expression level of a target gene in cells ortissues. The disease to be treated or prevented is not particularlylimited as long as it is a disease derived from the expression of atarget gene. The disease is preferably cancer, anemia, liver disease,gallbladder disease, fibrosis, or genetic disease. The nucleic acidlipid particle of the present invention can be administered to a mammal(preferably a human) in need thereof.

The present invention provides a method for inhibiting ordown-regulating the expression of a target gene in a cell or a tissue.When the target gene is non-coding RNA that is not translated into aprotein, the present invention also provides a method for inhibiting ordown-regulating the expression of the non-coding RNA and furtherup-regulating or, in some cases, down-regulating the expression of agene involved in the non-coding RNA.

EXAMPLES

Hereinafter, the present invention will be described more specificallywith reference to Examples, Reference Examples, and Test Examples.However, the present invention is not intended to be limited to them. Inthe Examples below, procedures of genetic engineering were performed bythe methods described in “Molecular Cloning” [Sambrook, J., Fritsch, E.F. and Maniatis, T., published in 1989 by Cold Spring Harbor LaboratoryPress] or according to the instructions of the commercially availablereagents or kits used, unless otherwise specified.

Reference Example 14-Nitrophenyl(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbonate

To a solution of diisopropylethylamine (0.50 g, 3.8 mmol) and4-dimethylaminopyridine (0.12 g, 0.95 mmol) in dichloromethane (61 mL),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.50 g, 0.95mmol) described in Examples 1 and 7 of WO2010042877 and 4-nitrophenylchloroformate (0.38 g, 1.9 mmol) were added, and the mixture was reactedat room temperature for 5 hours. Volatile matter was removed underreduced pressure, and a solid formed by the addition of ethyl acetatewas filtered off. The solvent in the obtained solution was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest (0.61 g, 93%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.8 Hz), 1.21-1.44 (36H, m),1.59-1.73 (4H, m), 2.00-2.09 (8H, m), 2.77 (4H, t, J=6.8 Hz), 4.77-4.85(1H, m), 5.28-5.42 (8H, m), 7.38 (2H, d, J=9.3 Hz), 8.28 (2H, d, J=9.3Hz).

Reference Example 22-(Dimethylamino)ethyl(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbonate

To a solution of4-nitrophenyl(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbonate (0.20 g, 0.29 mmol) obtained in Reference Example 1,2-dimethylaminoethanol (0.26 g, 2.9 mmol), and diisopropylethylamine(0.15 g, 1.2 mmol) in dichloromethane (10 mL), 4-dimethylaminopyridine(0.14 g, 1.2 mmol) was added, and the mixture was reacted overnight atroom temperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (100 mg, 54%). This compoundis a compound described in a table of WO2010/054405.

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.8 Hz), 1.20-1.40 (36H, m),1.45-1.65 (4H, m), 1.96-2.08 (8H, m), 2.28 (6H, s), 2.60 (2H, t, J=5.9Hz), 2.77 (4H, t, J=6.8 Hz), 4.21 (2H, t, J=5.9 Hz), 4.64-4.71, 1H, m),5.27-5.42 (8H, m).

MS (ESI+) m/z 644 [M+H]⁺

HRMS (ESI+) m/z 644.6012 (3.0 mDa).

Example 13-(Dimethylamino)propyl(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbonate (Exemplary Compound 1-467)

To a solution of4-nitrophenyl(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ylcarbonate(0.20 g, 0.29 mmol) obtained in Reference Example 1,3-dimethylamino-1-propanol (0.30 g, 2.9 mmol), and diisopropylethylamine(0.15 g, 1.2 mmol) in dichloromethane (10 mL), 4-dimethylaminopyridine(0.14 g, 1.2 mmol) was added, and the mixture was reacted overnight atroom temperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (100 mg, 53%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.8 Hz), 1.20-1.40 (36H, m),1.45-1.65 (4H, m), 1.84 (2H, tt, J=6.3, 7.3 Hz), 1.95-2.10 (8H, m), 2.22(6H, s), 2.36 (2H, t, J=7.3 Hz), 2.77 (4H, t, J=6.8 Hz), 4.18 (2H, t,J=6.3 Hz), 4.63-4.73, 1H, m), 5.27-5.43 (8H, m).

MS (ESI+) m/z 658 [M+H]⁺

HRMS (ESI+) m/z 658.6164 (2.6 mDa).

Reference Example 3(6Z,9Z,26Z,29Z)-Pentatriaconta-6,9,26,29-tetraen-18-one

To a solution of methyl linoleate (30.0 g, 101 mmol) in xylene (55 mL),a suspension of sodium hydride (5.05 g, 63%, 132 mmol) washed in advancewith hexane in xylene (10 mL) was added over 10 minutes, and the mixturewas then reacted at 150° C. for 5 hours. The reaction mixture was cooledto room temperature, then treated with water, and subjected toextraction with a hexane-ethyl acetate mixed solution. The obtainedorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was distilled off under reduced pressure to obtain an oil. Tothis oil, tetrahydrofuran (413 mL) and a 5 N aqueous sodium hydroxidesolution (102 mL) were added, and the mixture was reacted at 100° C. for5.5 hours. The reaction mixture was cooled to room temperature, thentreated with water, and subjected to extraction with a hexane-ethylacetate mixed solution. The obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(23.0 g, 91%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.6 Hz), 1.24-1.40 (28H, m),1.51-1.60 (4H, m), 2.01-2.09 (8H, m), 2.38 (4H, t, J=7.4 Hz), 2.77 (4H,t, J=6.6 Hz), 5.29-5.43 (8H, m).

Reference Example 4(6Z,9Z,26Z,29Z)-Pentatriaconta-6,9,26,29-tetraen-18-ol

To a solution of (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-one(23.0 g, 46.1 mmol) obtained in Reference Example 3 in methanol (187 mL)and tetrahydrofuran (187 mL), sodium borohydride (1.74 g, 46.1 mmol) wasadded, and the mixture was then reacted at room temperature for 80minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ahexane-ethyl acetate mixed solution, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (22.2 g, 96%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.24-1.48 (36H, m),2.01-2.09 (8H, m), 2.77 (4H, t, J=6.3 Hz), 3.55-3.62 (1H, m), 5.29-5.43(8H, m).

Reference Example 54-Nitrophenyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate

To a solution of diisopropylethylamine (0.20 g, 1.5 mmol) and4-dimethylaminopyridine (0.05 g, 0.38 mmol) in dichloromethane (25 mL),(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ol (0.19 g, 0.38mmol) obtained in Reference Example 4 and 4-nitrophenyl chloroformate(0.15 g, 0.77 mmol) were added, and the mixture was reacted overnight atroom temperature. Volatile matter was removed under reduced pressure,and a solid formed by the addition of hexane was filtered off. Thesolvent in the obtained solution was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest (0.12 g, 47%).

Example 23-(Dimethylamino)propyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate (Exemplary Compound 1-118)

To a solution of4-nitrophenyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate (0.03 g, 0.05 mmol) obtained in Reference Example 5,3-dimethylamino-1-propanol (0.05 g, 0.5 mmol), and diisopropylethylamine(0.03 g, 0.2 mmol) in dichloromethane (6 mL), 4-dimethylaminopyridine(0.02 g, 0.2 mmol) was added, and the mixture was reacted at roomtemperature for 4 days. 3-Dimethylamino-1-propanol (0.1 g, 1 mmol) wasfurther added thereto, and the mixture was reacted for additional 2days. After water treatment to terminate the reaction, the reactionmixture was subjected to extraction with dichloromethane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (10 mg, 31%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.6 Hz), 1.22-1.42 (34H, m),1.48-1.65 (4H, m), 1.85 (2H, tt, J=6.6, 7.4 Hz), 1.98-2.10 (8H, m), 2.22(6H, s), 2.36 (2H, t, J=7.4 Hz), 2.77 (4H, t, J=6.6 Hz), 4.18 (2H, t,6.6 Hz), 4.64-4.73 (1H, m), 5.37-5.43 (8H, m).

MS (ESI+) m/z 630 [M+H]⁺

HRMS (ESI+) m/z 630.5839 (1.4 mDa).

Example 34-(Dimethylamino)butyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate (Exemplary Compound 1-129)

To a solution of4-nitrophenyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate (0.12 g, 0.18 mmol) obtained in Reference Example 5,4-dimethylamino-1-butanol (0.22 g, 1.8 mmol), and diisopropylethylamine(0.10 g, 0.72 mmol) in dichloromethane (10 mL), 4-dimethylaminopyridine(0.09 g, 0.72 mmol) was added, and the mixture was reacted overnight atroom temperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (30 mg, 26%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.6 Hz), 1.21-1.42 (34H, m),1.47-1.64 (6H, m), 1.71 (2H, tt, J=6.6, 7.4 Hz), 1.99-2.09 (8H, m), 2.21(6H, s), 2.27 (2H, t, J=7.4 Hz), 2.77 (4H, t, J=6.6 Hz), 4.14 (2H, t,J=6.6 Hz), 4.63-4.72 (1H, m), 5.28-5.43 (8H, m).

MS (ESI+) m/z 644 [M+H]⁺

HRMS (ESI+) m/z 644.6008 (2.6 mDa).

Reference Example 6 (9Z,26Z)-Pentatriaconta-9,26-dien-18-one

To a solution of methyl oleate (10.0 g, 33.4 mmol) in xylene (18 mL), asuspension of sodium hydride (1.65 g, 63%, 43.4 mmol) washed in advancewith hexane in xylene (3 mL) was added over 5 minutes, and the mixturewas then reacted at 150° C. for 5 hours. The reaction mixture was cooledto room temperature, then treated with water, and subjected toextraction with a hexane-ethyl acetate mixed solution. The obtainedorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was distilled off under reduced pressure to obtain an oil. Tothis oil, tetrahydrofuran (135 mL) and a 5 N aqueous sodium hydroxidesolution (33 mL) were added, and the mixture was reacted at 100° C. for5.5 hours. The reaction mixture was cooled to room temperature, thentreated with water, and subjected to extraction with a hexane-ethylacetate mixed solution. The obtained organic layer was dried overanhydrous magnesium sulfate, and the solvent was distilled off underreduced pressure, followed by the formation of a solid with anacetone-hexane solvent. After removal of the solid by filtration, thesolvent was distilled off from the resulting solution under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(7.50 g, 89%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.8 Hz), 1.21-1.36 (40H, m),1.52-1.60 (4H, m), 1.97-2.04 (8H, m), 2.38 (4H, t, J=7.6 Hz), 5.30-5.39(4H, m).

Reference Example 7 (9Z,26Z)-Pentatriaconta-9,26-dien-18-ol

To a solution of (9Z,26Z)-pentatriaconta-9,26-dien-18-one (5.0 g, 9.9mmol) obtained in Reference Example 6 in methanol (40 mL) andtetrahydrofuran (40 mL), sodium borohydride (0.38 g, 9.9 mmol) wasadded, and the mixture was then reacted at room temperature for 80minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (4.5 g, 90%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (6H, t, J=6.6 Hz), 1.21-1.37 (48H, m),1.37-1.49 (4H, m), 1.97-2.06 (8H, m), 3.55-3.62 (1H, m), 5.31-5.40 (4H,m).

Example 4 3-Dimethylaminopropyl(9Z,26Z)-pentatriaconta-9,26-dien-18-ylcarbonate (Exemplary Compound 1-50)

To a solution of (9Z,26Z)-pentatriaconta-9,26-dien-18-ol (0.25 g, 0.50mmol) obtained in Reference Example 7 and pyridine (0.25 g, 3.1 mmol) intoluene (5.0 mL), a solution of triphosgene (0.10 g, 0.34 mmol) intoluene (0.74 mL) was added over 2 minutes. After stirring at roomtemperature for 100 minutes, 3-dimethylamino-1-propanol (0.54 g, 5.2mmol) was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (296 mg, 94%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.86-0.91 (6H, m), 1.22-1.36 (44H, m),1.50-1.59 (4H, m), 1.84 (2H, tt, J=6.8, 7.3 Hz), 1.97-2.04 (8H, m), 2.22(6H, s), 2.36 (2H, t, J=7.3 Hz), 4.18 (2H, t, J=6.8 Hz), 4.65-4.72 (1H,m), 5.33-5.37 (4H, m).

MS (ESI+) m/z 634 [M+H]⁺

HRMS (ESI+) m/z 634.6169 (3.1 mDa).

Reference Example 8(3Z,6Z,9Z,26Z,29Z,32Z)-Pentatriaconta-3,6,9,26,29,32-hexaen-18-one

To a solution of methyl α-linolenate (10.0 g, 33.4 mmol) in xylene (18mL), a suspension of sodium hydride (1.65 g, 63%, 43.4 mmol) washed inadvance with hexane in xylene (3 mL) was added over 5 minutes, and themixture was then reacted at 150° C. for 5 hours. The reaction mixturewas cooled to room temperature, then treated with water, and subjectedto extraction with a hexane-ethyl acetate mixed solution. The obtainedorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was distilled off under reduced pressure to obtain an oil. Tothis oil, tetrahydrofuran (135 mL) and a 5 N aqueous sodium hydroxidesolution (33 mL) were added, and the mixture was reacted at 100° C. for5.5 hours. The reaction mixture was cooled to room temperature, thentreated with water, and subjected to extraction with a hexane-ethylacetate mixed solution. The obtained organic layer was dried overanhydrous magnesium sulfate, and the solvent was distilled off underreduced pressure, followed by the formation of a solid with anacetone-hexane solvent. After removal of the solid by filtration, thesolvent was distilled off from the resulting solution under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(6.10 g, 74%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.98 (6H, t, J=7.6 Hz), 1.22-1.40 (16H, m),1.52-1.60 (4H, m), 2.00-2.19 (8H, m), 2.39 (4H, t, J=7.6 Hz), 2.74-2.83(8H, m), 5.28-5.43 (12H, m).

Reference Example 9(3Z,6Z,9Z,26Z,29Z,32Z)-Pentatriaconta-3,6,9,26,29,32-hexaen-18-ol

To a solution of(3Z,6Z,9Z,26Z,29Z,32Z)-pentatriaconta-3,6,9,26,29,32-hexaen-18-one (5.0g, 10.1 mmol) obtained in Reference Example 8 in methanol (41 mL) andtetrahydrofuran (41 mL), sodium borohydride (0.38 g, 10 mmol) was added,and the mixture was then reacted at room temperature for 80 minutes.After treatment with a saturated aqueous solution of ammonium chloride,the reaction mixture was subjected to extraction with ethyl acetate, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (4.0 g, 79%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.98 (6H, t, J=7.6 Hz), 1.24-1.48 (24H, m),2.00-2.20 (8H, m), 2.75-2.84 (8H, m), 3.55-3.61 (1H, m), 5.29-5.43 (12H,m).

Example 53-Dimethylaminopropyl(3Z,6Z,9Z,26Z,29Z,32Z)-pentatriaconta-3,6,9,26,29,32-hexaen-18-ylcarbonate (Exemplary Compound 1-176)

To a solution of(3Z,6Z,9Z,26Z,29Z,32Z)-pentatriaconta-3,6,9,26,29,32-hexaen-18-ol (0.25g, 0.50 mmol) obtained in Reference Example 9 and pyridine (0.25 g, 3.1mmol) in toluene (5.0 mL), a solution of triphosgene (0.10 g, 0.34 mmol)in toluene (0.75 mL) was added over 2 minutes. After stirring at roomtemperature for 100 minutes, 3-dimethylamino-1-propanol (0.55 g, 5.3mmol) was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (274 mg, 87%,isomeric mixture).

¹H-NMR (500 MHz, CDCl₃) δ: 0.98 (6H, t, J=7.6 Hz), 1.24-1.39 (20H, m),1.49-1.62 (4H, m), 1.85 (2H, tt, J=6.6, 7.6 Hz), 2.00-2.19 (8H, m),2.22, (6H, s), 2.36 (2H, t, J=7.6 Hz), 2.74-2.84 (8H, m), 4.17 (2H, t,J=6.6 Hz), 4.65-4.71 (1H, m), 5.28-5.44 (12H, m).

MS (ESI+) m/z 626 [M+H]⁺

HRMS (ESI+) m/z 626.5512 (−0.5 mDa).

Reference Example 10(6Z,9Z,12Z,23Z,26Z,29Z)-Pentatriaconta-6,9,12,23,26,29-hexaen-18-one

To a solution of methyl γ-linolenate (5.00 g, 17.1 mmol) in xylene (9mL), a suspension of sodium hydride (0.85 g, 63%, 22.2 mmol) washed inadvance with hexane in xylene (1.5 mL) was added over 5 minutes, and themixture was then reacted at 150° C. for 5 hours. The reaction mixturewas cooled to room temperature, then treated with water, and subjectedto extraction with a hexane-ethyl acetate mixed solution. The obtainedorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was distilled off under reduced pressure to obtain an oil. Tothis oil, tetrahydrofuran (69 mL) and a 5 N aqueous sodium hydroxidesolution (17 mL) were added, and the mixture was reacted at 100° C. for5.5 hours. The reaction mixture was cooled to room temperature, thentreated with water, and subjected to extraction with a hexane-ethylacetate mixed solution. The obtained organic layer was dried overanhydrous magnesium sulfate, and the solvent was distilled off underreduced pressure, followed by the formation of a solid with anacetone-hexane solvent. After removal of the solid by filtration, thesolvent was distilled off from the resulting solution under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(3.80 g, 90%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.86-0.91 (6H, m), 1.23-1.42 (16H, m),1.55-1.63 (4H, m), 1.98-2.20 (8H, m), 2.39 (4H, t, J=7.6 Hz), 2.78-2.83(8H, m), 5.25-5.44 (12H, m).

Reference Example 11(6Z,9Z,12Z,23Z,26Z,29Z)-Pentatriaconta-6,9,12,23,26,29-hexaen-18-ol

To a solution of(6Z,9Z,12Z,23Z,26Z,29Z)-pentatriaconta-6,9,12,23,26,29-hexaen-18-one(3.0 g, 6.1 mmol) obtained in Reference Example 10 in methanol (25 mL)and tetrahydrofuran (25 mL), sodium borohydride (0.23 g, 6.1 mmol) wasadded, and the mixture was then reacted at room temperature for 80minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (2.1 g, 70%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.86-0.91 (6H, m), 1.24-1.50 (24H, m),1.99-2.21 (8H, m), 2.78-2.84 (8H, m), 3.55-3.62 (1H, m), 5.28-5.44 (12H,m).

Example 63-Dimethylaminopropyl(6Z,9Z,12Z,23Z,26Z,29Z)-pentatriaconta-6,9,12,23,26,29-hexaen-18-ylcarbonate (Exemplary Compound 1-152)

To a solution of(6Z,9Z,12Z,23Z,26Z,29Z)-pentatriaconta-6,9,12,23,26,29-hexaen-18-ol(0.25 g, 0.50 mmol) obtained in Reference Example 11 and pyridine (0.25g, 3.1 mmol) in toluene (5.0 mL), a solution of triphosgene (0.10 g,0.34 mmol) in toluene (0.75 mL) was added over 2 minutes. After stirringat room temperature for 100 minutes, 3-dimethylamino-1-propanol (0.55 g,5.3 mmol) was added thereto, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (240 mg, 76%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.86-0.91 (6H, m), 1.22-1.42 (20H, m),1.51-1.63 (4H, m), 1.85 (2H, tt, J=6.6, 7.3 Hz), 1.99-2.19 (8H, m), 2.22(6H, s), 2.36 (2H, t, J=7.3 Hz), 2.78-2.83 (8H, m), 4.17 (2H, t, J=6.6Hz), 4.65-4.72 (1H, m), 5.30-5.45 (12H, m).

MS (ESI+) m/z 626 [M+H]⁺

HRMS (ESI+) m/z 626.5515 (0.3 mDa).

Reference Example 12 Dimethyldi-(9Z,12Z)-octadeca-9,12-dien-1-ylpropanedioate

To a solution of dimethyl malonate (13.5 g, 102 mmol) in toluene (500mL), a suspension of sodium hydride (5.17 g, 63%, 136 mmol) washed inadvance with hexane in toluene (6 mL) was added over 5 minutes. Afterstirring at 80° C. for 30 minutes,(9Z,12Z)-octadeca-9,12-dien-1-ylmethanesulfonate (compound described inExample 1 of WO2009/132131, 23.4 g, 67.9 mmol) was added thereto, andthe mixture was stirred at 100° C. for 4 hours and stirred at 120° C.for 2 hours. After treatment with a 1 N aqueous hydrochloric acidsolution, the reaction mixture was subjected to extraction, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (9.71 g).

¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (6H, t, J=7.0 Hz), 1.07-1.16 (4H, m),1.21-1.40 (32H, m), 1.82-1.89 (4H, m), 2.01-2.08 (8H, m), 2.77 (4H, t,J=6.6 Hz), 3.71 (6H, s), 5.29-5.43 (8H, m).

A by-product dimethyl (9Z,12Z)-octadeca-9,12-dien-1-ylpropanedioate wasalso obtained as a colorless liquid (8.50 g).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85 (3H, t, J=7.0 Hz), 1.21-1.37 (18H, m),1.81-1.90 (2H, m), 1.96-2.05 (4H, m), 2.74 (2H, t, J=6.6 Hz), 3.32 (1H,t, J=7.4 Hz), 3.70 (6H, s), 5.25-5.39 (4H, m).

Reference Example 13 Methyl(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11-dienoate

To a solution of dimethyldi-(9Z,12Z)-octadeca-9,12-dien-1-ylpropanedioate (5.00 g, 7.95 mmol)obtained in Reference Example 12 and water (2.15 g, 119 mmol) indimethyl sulfoxide (39.5 mL), lithium chloride (1.01 g, 23.9 mmol) wasadded. After stirring at 150° C. for 5 hours, water (2.15 g, 119 mmol)and lithium chloride (1.01 g, 23.9 mmol) were added. The mixture wasreacted at 160° C. for 5 hours and cooled to room temperature. Afterwater treatment to terminate the reaction, the reaction mixture wassubjected to extraction with hexane, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (3.00 g, 66%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.20-1.47 (38H, m),1.55-1.64 (2H, m), 2.01-2.09 (8H, m), 2.28-2.37 (1H, m), 2.77 (4H, t,J=6.6 Hz), 3.67 (3H, s), 5.29-5.46 (8H, m).

Reference Example 14(11Z,14Z)-2-[(9Z,12Z)-Octadeca-9,12-dien-1-yl]icosa-11,14-dien-1-ol

To a solution of methyl(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11-dienoate (3.00 g,5.25 mmol) obtained in Reference Example 13 in tetrahydrofuran (40 mL),lithium aluminum hydride (0.400 g, 10.5 mmol) was added, and the mixturewas stirred at room temperature for 1 hour. The reaction mixture wastreated with water (0.4 mL), a 15% aqueous sodium hydroxide solution(0.4 mL), and water (1.2 mL) and subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (1.00 g, 35%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.24-1.44 (38H, m),1.97-2.09 (9H, m), 2.78 (4H, t, J=6.6 Hz), 4.15 (2H, t, J=6.3 Hz),5.29-5.43 (8H, m).

Example 73-(Dimethylamino)propyl(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11,14-dien-1-ylcarbonate (Exemplary Compound 1-477)

A solution of(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11,14-dien-1-ol(0.15 g, 0.28 mmol) obtained in Reference Example 14 and pyridine (0.14g, 1.8 mmol) in toluene (0.6 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.06 g, 0.19 mmol) in toluene (0.24 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 2 hours, thereaction mixture was heated to 10° C., stirred for 30 minutes, andcooled to 0° C. again. 3-Dimethylamino 1-propanol (0.30 g, 2.9 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (130 mg, 70%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.21-1.40 (40H, m),1.62-1.69 (1H, m), 1.85 (2H, tt, J=6.6, 7.4 Hz), 2.01-2.09 (8H, m), 2.22(6H, s), 2.36 (2H, t, J=7.4 Hz), 2.78 (4H, t, J=6.6 Hz), 4.03 (2H, d,J=5.9 Hz), 4.18 (2H, t, J=6.6 Hz), 5.29-5.43 (8H, m).

MS (ESI+) m/z 672 [M+H]⁺

HRMS (ESI+) m/z 672.6309 (1.4 mDa).

Reference Example 15 (9Z,12Z)-N-Methoxy-N-methyloctadeca-9,12-dienamide

To a solution of linoleic acid (10.0 g, 35.7 mmol) andN,O-dimethylhydroxylamine hydrochloride (6.56 g, 71.3 mmol) indichloromethane (250 mL), 1-hydroxybenzimidazole hydrate (10.9 g, 71.3mmol), triethylamine (7.22 g, 71.3 mmol), and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (13.7 g,71.3 mmol) were added, and the mixture was reacted overnight at roomtemperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (11.4 g, 99%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 1.24-1.40 (14H, m),1.63 (2H, quint, J=7.4 Hz), 2.00-2.09 (4H, m), 2.41 (2H, t, J=7.4 Hz),2.77 (2H, t, J=6.6 Hz), 3.18 (3H, s), 3.68 (3H, s), 5.29-5.43 (4H, m).

Reference Example 16 (19Z,22Z)-Octacosa-19,22-dien-11-one

A solution of (9Z,12Z)-N-methoxy-N-methyloctadeca-9,12-dienamide (11.4g, 35.2 mmol) obtained in Reference Example 15 in tetrahydrofuran (157mL) was cooled to 15° C. in a water bath. A solution of 1 N n-decylmagnesium bromide in tetrahydrofuran (52.9 mL, 52.9 mmol) was addeddropwise thereto over 20 minutes, and the mixture was then reactedovernight at room temperature. After treatment with a saturated aqueoussolution of ammonium chloride, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(14.3 g, 99%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 0.90 (3H, t, J=7.0Hz), 1.20-1.40 (28H, m), 1.50-1.60 (4H, m), 2.00-2.09 (4H, m), 2.38 (4H,t, J=7.4 Hz), 2.77 (2H, t, J=6.6 Hz), 5.28-5.42 (4H, m).

Reference Example 17 (19Z,22Z)-Octacosa-19,22-dien-11-ol

To a solution of (19Z,22Z)-octacosa-19,22-dien-11-one (14.3 g, 35.2mmol) obtained in Reference Example 16 in methanol (106 mL) andtetrahydrofuran (106 mL), sodium borohydride (1.33 g, 35.2 mmol) wasadded, and the mixture was then reacted at room temperature for 70minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with hexane,and the obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtainthe compound of interest as a colorless liquid (13.5 g, 95%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 0.90 (3H, t, J=7.0Hz), 1.23-1.47 (40H, m), 2.01-2.09 (4H, m), 2.77 (2H, t, J=6.6 Hz),3.54-3.62 (1H, m), 5.29-5.42 (4H, m).

Example 8 3-Dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 1-72)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.15 g, 0.37 mmol)obtained in Reference Example 17 and pyridine (0.18 g, 2.3 mmol) intoluene (0.8 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.07 g, 0.25 mmol) in toluene (0.31 mL) was added theretoover 2 minutes. After stirring at 0° C. for 1 hour, the reaction mixturewas heated to 10° C., stirred for 20 minutes, and cooled to 0° C. again.3-Dimethylamino 1-propanol (0.40 g, 3.9 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (100 mg, 51%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.22-1.39 (32H, m),1.49-1.62 (4H, m), 1.85 (2H, tt, J=6.6, 7.6 Hz), 2.01-2.08 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.6 Hz), 2.77 (2H, t, J=6.6 Hz), 4.18 (2H, t,J=6.6 Hz), 4.65-4.72 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 536 [M+H]⁺

HRMS (ESI+) m/z 536.5038 (−0.5 mDa).

Example 9(1-Methylpiperidin-3-yl)methyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-72)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.26 g, 0.64 mmol)obtained in Reference Example 17 and pyridine (0.32 g, 4.0 mmol) intoluene (7.4 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.13 g, 0.44 mmol) in toluene (0.9 mL) was added theretoover 2 minutes. After stirring at 0° C. for 20 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. (1-Methyl-3-piperidyl)methanol (0.87 g, 6.7 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (360 mg, 55%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.21-1.39 (32H, m),1.49-1.76 (9H, m), 1.86-1.92 (1H, m), 1.96-2.07 (5H, m), 2.26 (3H, s),2.74 (1H, d, J=10.5 Hz), 2.77 (2H, t, J=6.8 Hz), 2.85 (1H, d, J=10.5Hz), 3.94 (1H, dt, J=3.2, 7.3 Hz), 4.06 (1H, ddd, J=3.2, 5.9, 10.7 Hz),4.65-4.71 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 562 [M+H]⁺

HRMS (ESI+) m/z 562.5196 (−0.3 mDa).

Example 10 1-Methylpiperidin-4-yl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-66)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.300 g, 0.738 mmol)obtained in Reference Example 17 and pyridine (0.368 g, 4.65 mmol) intoluene (7.4 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.151 g, 0.509 mmol) in toluene (1.1 mL) was added theretoover 2 minutes. After stirring at 0° C. for 20 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. 4-Hydroxy-1-methylpiperidine (0.892 g, 7.75 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (249 mg, 62%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.20-1.40 (32H, m),1.49-1.62 (4H, m), 1.73-1.84 (2H, m), 1.92-2.01 (2H, m), 2.01-2.08 (4H,m), 2.16-2.25 (2H, m), 2.28 (3H, s), 2.65-2.73 (2H, m), 2.77 (2H, t,J=6.6 Hz), 4.57-4.66 (1H, m), 4.64-4.73 (1H, m), 5.28-5.43 (4H, m).

MS (ESI+) m/z 548 [M+H]⁺

HRMS (ESI+) m/z 548.5042 (−0.1 mDa).

Example 11(1-Methylpyrrolidin-3-yl)methyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-71)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.300 g, 0.738 mmol)obtained in Reference Example 17 and pyridine (0.368 g, 4.65 mmol) intoluene (7.4 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.151 g, 0.509 mmol) in toluene (1.1 mL) was added theretoover 2 minutes. After stirring at 0° C. for 20 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. (1-Methylpyrrolidin-3-yl)methanol (0.892 g, 7.75 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (234 mg, 58%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.20-1.40 (32H, m),1.45-1.64 (5H, m), 1.95-2.07 (5H, m), 2.30 (1H, dd, J=5.5, 9.4 Hz), 2.34(3H, s), 2.51 (2H, t, J=7.0 Hz), 2.23-2.62 (1H, m), 2.65 (1H, dd, J=7.8,9.0 Hz), 2.77 (2H, t, J=6.3 Hz), 4.03 (1H, ddd, J=2.0, 7.8, 9.8 Hz),4.07 (1H, ddd, J=2.0, 7.0, 10.6 Hz), 4.68 (1H, tt, J=5.5, 7.0 Hz),5.29-5.43 (4H, m).

MS (ESI+) m/z 548 [M+H]⁺

HRMS (ESI+) m/z 548.5050 (0.7 mDa).

Example 12 1-Methylpyrrolidin-3-yl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-64)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.150 g, 0.369 mmol)obtained in Reference Example 17 and pyridine (0.184 g, 2.32 mmol) intoluene (3.7 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.0755 g, 0.254 mmol) in toluene (0.55 mL) was addedthereto over 2 minutes. After stirring at 0° C. for 20 minutes, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 1-Methyl-3-pyrrolidinol (0.392 g, 3.87 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (99.9 mg, 50%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 0.89 (3H, t, J=7.0Hz), 1.22-1.40 (32H, m), 1.47-1.60 (4H, m), 1.92 (1H, dddd, J=2.7, 6.3,7.4, 13.7 Hz), 2.01-2.09 (4H, m), 2.28 (1H, dddd, J=6.3, 7.4, 7.8, 13.7Hz), 2.36 (3H, s), 2.41 (1H, ddd, J=6.3, 7.8, 9.0 Hz), 2.67 (1H, dd,J=2.7, 10.9 Hz), 2.73 (1H, ddd, J=6.3, 7.4, 9.0 Hz), 2.77 (2H, t, J=6.6Hz), 2.82 (1H, dd, J=5.9, 10.9 Hz), 4.67 (1H, tt, J=5.5, 7.0 Hz), 5.08(1H, ddt, J=5.9, 7.8, 2.7 Hz), 5.28-5.43 (4H, m).

MS (ESI+) m/z 534 [M+H]⁺

HRMS (ESI+) m/z 534.4891 (0.5 mDa).

Example 132-(1-Methylpyrrolidin-2-yl)ethyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-75)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.200 g, 0.492 mmol)obtained in Reference Example 17 and pyridine (0.245 g, 3.10 mmol) intoluene (5 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.101 g, 0.339 mmol) in toluene (0.74 mL) was added theretoover 2 minutes. After stirring at 0° C. for 15 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. 1-Methyl-2-pyrrolidinemethanol (0.667 g, 5.16 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (98.5 mg, 36%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 0.89 (3H, t, J=7.0Hz), 1.22-1.40 (32H, m), 1.44-1.85 (8H, m), 1.92-2.18 (8H, m), 2.32 (3H,s), 2.77 (2H, t, J=6.6 Hz), 3.06 (1H, ddd, J=2.3, 8.2, 8.6 Hz),4.11-4.26 (2H, m), 4.65-4.72 (1H, m), 5.29-5.43 (4H, m).

MS (ESI+) m/z 562 [M+H]⁺

HRMS (ESI+) m/z 562.5203 (0.4 mDa).

Example 14(1-Methylpiperidin-4-yl)methyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 2-73)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.200 g, 0.492 mmol)obtained in Reference Example 17 and pyridine (0.245 g, 3.10 mmol) intoluene (5 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.101 g, 0.339 mmol) in toluene (0.74 mL) was added theretoover 2 minutes. After stirring at 0° C. for 15 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. 4-Hydroxymethyl-1-methylpiperidine (0.667 g, 5.16 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (21.7 mg, 8%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 0.90 (3H, t, J=7.0Hz), 1.23-1.39 (28H, m), 1.49-1.61 (6H, m), 1.63-1.71 (1H, m), 1.74 (2H,d, J=14.1 Hz), 1.91 (2H, t, J=11.7 Hz), 2.00-2.03 (4H, m), 2.27 (3H, s),2.77 (2H, t, J=6.6 Hz), 2.86 (2H, d, J=11.7 Hz), 3.98 (2H, d, J=6.6 Hz),4.64-4.71 (1H, m), 5.29-5.43 (4H, m).

MS (ESI+) m/z 562 [M+H]⁺

HRMS (ESI+) m/z 562.5204 (0.5 mDa).

Reference Example 18 (21Z,24Z)-Triaconta-21,24-dien-13-ol

To a solution of (9Z,12Z)-N-methoxy-N-methyloctacosa-9,12-dienamide(0.50 g, 1.5 mmol) obtained in Reference Example 15 in tetrahydrofuran(6.9 mL), a solution of 1 N n-dodecyl magnesium bromide in diethyl ether(4.6 mL, 4.6 mmol) was added dropwise over 3 minutes, and the mixturewas then reacted at room temperature for 6 hours. After treatment with asaturated aqueous solution of ammonium chloride, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain a mixture containing a presumedintermediate ketone (0.93 g). To a solution of this ketone mixture inmethanol (4.6 mL) and tetrahydrofuran (4.6 mL), sodium borohydride (0.06g, 1.5 mmol) was added, and the mixture was then reacted at roomtemperature for 3 hours. After treatment with a saturated aqueoussolution of ammonium chloride, the reaction mixture was subjected toextraction with ethyl acetate, and the obtained organic layer was driedover anhydrous magnesium sulfate. The solvent was distilled off underreduced pressure to obtain the compound of interest as a colorlessliquid (0.59 g, 89%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=6.8 Hz), 0.90 (3H, t, J=6.8Hz), 1.22-1.48 (44H, m), 2.02-2.08 (4H, m), 2.77 (2H, t, J=6.8 Hz),3.55-3.62 (1H, m), 5.30-5.42 (4H, m).

Example 15 3-(Dimethylamino)propyl(21Z,24Z)-triaconta-21,24-dien-13-ylcarbonate (Exemplary Compound 1-112)

A solution of (21Z,24Z)-triaconta-21,24-dien-13-ol (0.15 g, 0.35 mmol)obtained in Reference Example 18 and pyridine (0.17 g, 2.2 mmol) intoluene (3.4 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.07 g, 0.24 mmol) in toluene (0.5 mL) was added theretoover 1 minute. After stirring at 0° C. for 2 hours, 3-dimethylamino1-propanol (0.37 g, 3.6 mmol) was added thereto, and the mixture wasreacted overnight at room temperature. After treatment with a saturatedaqueous solution of sodium bicarbonate, the reaction mixture wassubjected to extraction with hexane, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (177 mg, 91%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.21-1.40 (36H, m),1.49-1.63 (4H, m), 1.84 (2H, tt, J=6.6, 7.6 Hz), 2.01-2.08 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.6 Hz), 2.78 (2H, t, J=6.6 Hz), 4.18 (2H, t,J=6.6 Hz), 4.65-4.72 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 564 [M+H]⁺

HRMS (ESI+) m/z 564.5359 (0.3 mDa).

Reference Example 19 (19Z,22Z)-Octacosa-19,22-dien-3-yn-11-one

To a solution of (9Z,12Z)-N-methoxy-N-methyl-octacosa-9,12-dienamide(1.00 g, 3.09 mmol) obtained in Reference Example 15 in tetrahydrofuran(6.2 mL), a solution of 0.5 N (decyn-7-ynyl)magnesium chloride intetrahydrofuran (12.4 mL, 6.20 mmol) was added dropwise over 3 minutes,and the mixture was then reacted at room temperature for 6 hours. Aftertreatment with a saturated aqueous solution of ammonium chloride, thereaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (1.03 g, 83%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.1 Hz), 1.11 (3H, t, J=7.3Hz), 1.23-1.61 (24H, m), 2.01-2.08 (4H, m), 2.11-2.19 (4H, m), 2.36-2.41(4H, m), 2.77 (2H, t, J=6.8 Hz), 5.29-5.42 (4H, m).

Reference Example 20 (19Z,22Z)-Octacosa-19,22-dien-3-yn-11-ol

To a solution of (19Z,22Z)-octacosa-19,22-dien-3-yn-11-one (1.0 g, 2.56mmol) obtained in Reference Example 19 in methanol (7.7 mL) andtetrahydrofuran (7.7 mL), sodium borohydride (0.097 g, 2.6 mmol) wasadded, and the mixture was then reacted at room temperature for 3 hours.After treatment with a saturated aqueous solution of ammonium chloride,the reaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (0.78 g, 75%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.1 Hz), 1.11 (3H, t, J=7.3Hz), 1.24-1.52 (28H, m), 2.02-2.08 (4H, m), 2.11-2.19 (4H, m), 2.77 (2H,t, J=6.8 Hz), 3.55-3.62 (1H, m), 5.30-5.42 (4H, m).

Example 163-(Dimethylamino)propyl(19Z,22Z)-octacosa-19,22-dien-3-yn-11-ylcarbonate (Exemplary Compound 1-99)

A solution of (19Z,22Z)-octacosa-19,22-dien-3-yn-11-ol (0.15 g, 0.37mmol) obtained in Reference Example 20 and pyridine (0.19 g, 2.4 mmol)in toluene (3.7 mL) was cooled to 0° C. in an ice bath, and a solutionof triphosgene (0.076 g, 0.26 mmol) in toluene (0.5 mL) was addedthereto over 1 minute. After stirring at 0° C. for 2 hours,3-dimethylamino 1-propanol (0.40 g, 3.9 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (183 mg, 93%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (3H, t, J=6.6 Hz), 1.11 (3H, t, J=7.3Hz), 1.23-1.64 (28H, m), 1.85 (2H, tt, J=6.6, 7.3 Hz), 2.01-2.08 (4H,m), 2.10-2.19 (4H, m), 2.22 (6H, s), 2.36 (2H, t, J=7.3 Hz), 2.77 (2H,t, J=6.6 Hz), 4.18 (2H, t, J=6.6 Hz), 4.64-4.71 (1H, m), 5.29-5.42 (4H,m).

MS (ESI+) m/z 532 [M+H]⁺

HRMS (ESI+) m/z 532.4739 (0.9 mDa).

Reference Example 21 (3Z,19Z,22Z)-Octacosa-3,19,22-trien-11-ol

To nickel(II) acetate tetrahydrate (0.24 g, 0.97 mmol), ethanol (12 mL)was added under the hydrogen gas atmosphere, and a solution of sodiumborohydride (0.037 g, 0.97 mmol) in ethanol (6 mL) was added. Afterstirring at room temperature for 15 minutes, ethylenediamine (0.23 g,3.9 mmol) was added, and the mixture was further stirred for 15 minutes.Subsequently, a solution of (19Z,22Z)-octacosa-19,22-dien-3-yn-11-ol(0.39 g, 0.97 mmol) obtained in Reference Example 20 in ethanol (6 mL)was added thereto over 1 minute, and the mixture was stirred at roomtemperature for 5.5 hours under the hydrogen atmosphere. The reactionmixture was diluted with a 20% solution of ethyl acetate in hexane andsubjected to silica gel column chromatography to obtain the compound ofinterest as a colorless liquid (0.37 g, 95%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.1 Hz), 0.95 (3H, t, J=7.3Hz), 1.23-1.48 (28H, m), 1.99-2.08 (8H, m), 2.77 (2H, t, J=6.8 Hz),3.55-3.61 (1H, m), 5.29-5.42 (6H, m).

Example 173-(Dimethylamino)propyl(3Z,19Z,22Z)-octacosa-3,19,22-trien-11-ylcarbonate (Exemplary Compound 1-87)

A solution of (3Z,19Z,22Z)-octacosa-3,19,22-trien-11-ol (0.15 g, 0.37mmol) obtained in Reference Example 21 and pyridine (0.19 g, 2.4 mmol)in toluene (3.7 mL) was cooled to 0° C. in an ice bath, and a solutionof triphosgene (0.075 g, 0.26 mmol) in toluene (0.5 mL) was addedthereto over 1 minute. After stirring at 0° C. for 2 hours,3-dimethylamino 1-propanol (0.40 g, 3.9 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (136 mg, 69%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.89 (3H, t, J=6.6 Hz), 0.95 (3H, t, J=7.6Hz), 1.22-1.40 (24H, m), 1.49-1.64 (4H, m), 1.84 (2H, tt, J=6.6, 7.3Hz), 1.97-2.08 (8H, m), 2.22 (6H, s), 2.36 (2H, t, J=7.3 Hz), 2.77 (2H,t, J=6.6 Hz), 4.17 (2H, J=6.6 Hz), 4.65-4.71 (1H, m), 5.27-5.43 (6H, m).

MS (ESI+) m/z 534 [M+H]⁺

HRMS (ESI+) m/z 534.4891 (0.5 mDa).

Example 184-(Dimethylamino)butyl(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11,14-dien-1-ylcarbonate (Exemplary Compound 1-478)

A solution of(11Z,14Z)-2-[(9Z,12Z)-octadeca-9,12-dien-1-yl]icosa-11,14-dien-1-ol(0.15 g, 0.28 mmol) obtained in Reference Example 14 and pyridine (0.14g, 1.8 mmol) in toluene (0.6 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.06 g, 0.19 mmol) in toluene (0.24 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 2 hours, thereaction mixture was heated to 10° C., stirred for 30 minutes, andcooled to 0° C. again. 4-Dimethylamino 1-butanol (0.35 g, 2.9 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (80 mg, 42%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.86-0.92 (6H, m), 1.20-1.40 (40H, m),1.50-1.59 (2H, m), 1.63-1.74 (3H, m), 2.01-2.09 (8H, m), 2.21 (6H, s),2.28 (2H, t, J=7.4 Hz), 2.78 (4H, t, J=6.6 Hz), 4.02 (2H, d, J=5.9 Hz),4.14 (2H, t, J=6.6 Hz), 5.29-5.43 (8H, m).

MS (ESI+) m/z 686 [M+H]⁺

HRMS (ESI+) m/z 686.6461 (1.0 mDa).

Reference Example 22 Octacosan-11-ol

To a solution of octadecanal (0.62 g) in tetrahydrofuran (2.3 mL), asolution of 1 N n-decyl magnesium bromide in diethyl ether (4.6 mL, 4.6mmol) was added, and the mixture was reacted at room temperature for 20minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with hexane,and the obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtainthe compound of interest as a white waxy solid (0.34 g, 36%).

Example 19 3-(Dimethylamino)propyloctacosan-11-yl carbonate (ExemplaryCompound 1-8)

A solution of octacosan-11-ol (0.11 g, 0.27 mmol) obtained in ReferenceExample 22 and pyridine (0.13 g, 1.7 mmol) in toluene (2.7 mL) wascooled to 0° C. in an ice bath, and a solution of triphosgene (0.055 g,0.18 mmol) in toluene (0.40 mL) was added thereto over 1 minute. Afterstirring at 0° C. for 15 minutes, the reaction mixture was heated toroom temperature, stirred for 1 hour, and cooled to 0° C. again.3-Dimethylamino-1-propanol (0.29 g, 2.8 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (114 mg, 79%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.35 (46H, m),1.48-1.64 (4H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 2.22 (6H, s), 2.36 (2H,t, J=7.4 Hz), 4.17 (2H, t, J=6.6 Hz), 4.64-4.72 (1H, m).

MS (ESI+) m/z 540 [M+H]⁺

HRMS (ESI+)m/z540.5344 (−1.2 mDa).

Reference Example 23 (Z)-N-Methoxy-N-methyloctadec-9-enamide

To a solution of oleic acid (10.3 g, 36.3 mmol) andN,O-dimethylhydroxylamine hydrochloride (7.08 g, 72.6 mmol) indichloromethane (250 mL), 1-hydroxybenzimidazole hydrate (11.1 g, 72.6mmol), triethylamine (7.34 g, 72.6 mmol), and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (13.9 g,72.6 mmol) were added, and the mixture was reacted overnight at roomtemperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (12.1 g, 99%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.22-1.37 (20H, m),1.63 (2H, quint, J=7.3 Hz), 1.97-2.04 (4H, m), 2.41 (2H, t, J=7.3 Hz),3.18 (3H, s), 3.68 (3H, s), 5.31-5.38 (2H, m).

Reference Example 24 (Z)-Octacos-19-en-11-one

To a solution of (Z)-N-methoxy-N-methyloctadec-9-enamide (0.50 g, 1.5mmol) obtained in Reference Example 23 in tetrahydrofuran (7.7 mL), asolution of 1 N n-decyl magnesium bromide in tetrahydrofuran (3.1 mL,3.1 mmol) was added, and the mixture was then reacted at roomtemperature for 1 hour and subsequently at 60° C. for 30 minutes. Aftertreatment with a saturated aqueous solution of ammonium chloride,volatile matter was removed under reduced pressure. The residue wassubjected to extraction with hexane, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (0.59 g, 93%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.90 (6H, m), 1.20-1.36 (34H, m),1.52-1.60 (4H, m), 1.98-2.04 (4H, m), 2.38 (4H, t, J=7.3 Hz), 5.32-5.38(2H, m).

Reference Example 25 (Z)-Octacos-19-en-11-ol

To a solution of (Z)-octacosa-19-en-11-one (0.59 g, 1.4 mmol) obtainedin Reference Example 24 in methanol (4.3 mL) and tetrahydrofuran (4.3mL), sodium borohydride (0.054 g, 1.4 mmol) was added, and the mixturewas then reacted at room temperature for 60 minutes. After treatmentwith a saturated aqueous solution of ammonium chloride, the reactionmixture was subjected to extraction with ethyl acetate, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain the compound ofinterest as a colorless liquid (0.45 g, 77%).

Example 20 3-(Dimethylamino)propyl(19Z)-octacos-19-en-11-yl carbonate(Exemplary Compound 1-16)

A solution of (Z)-octacosa-19-en-11-ol (0.45 g, 1.1 mmol) obtained inReference Example 25 and pyridine (0.55 g, 6.9 mmol) in toluene (11 mL)was cooled to 0° C. in an ice bath, and a solution of triphosgene (0.23g, 6.9 mmol) in toluene (1.7 mL) was added thereto over 2 minutes. Afterstirring at 0° C. for 30 minutes, the reaction mixture was heated toroom temperature, stirred for 1 hour, and cooled to 0° C. again.3-Dimethylamino-1-propanol (1.2 g, 12 mmol) was added thereto, and themixture was reacted at room temperature for 3 hours. After treatmentwith a saturated aqueous solution of sodium bicarbonate, the reactionmixture was subjected to extraction with hexane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain the compound ofinterest as a colorless liquid (530 mg, 89%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (6H, t, J=6.8 Hz), 1.21-1.37 (38H, m),1.49-1.62 (4H, m), 1.84 (2H, tt, J=6.6, 7.6 Hz), 1.97-2.04 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.6 Hz), 4.18 (2H, t, J=6.6 Hz), 4.65-4.71 (1H,m), 5.32-5.37 (2H, m).

MS (ESI+) m/z 538 [M+H]⁺

HRMS (ESI+) m/z 538.5193 (−0.6 mDa).

Reference Example 26 Octadeca-9,12,15-triyn-1-ol

To a solution of 9-decyn-1-ol (5.15 g, 33.4 mmol) and1-bromoocta-2,5-diyne (known compound, 6.18 g, 33.4 mmol) inN,N-dimethylformamide (66 mL), sodium iodide (5.56 g, 37.1 mmol),potassium carbonate (10.2 g, 74.1 mmol), and copper(I) iodide (7.06 g,37.1 mmol) were added in this order, and the mixture was reactedovernight at room temperature. Insoluble matter was removed throughcelite. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction with ahexane-ethyl acetate mixed solvent, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (8.40, 97%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.12 (3H, t, J=7.6 Hz), 1.24-1.42 (10H, m),1.48 (2H, tt, J=7.1, 7.6 Hz), 1.57 (2H, tt, J=6.6, 7.1 Hz), 2.12-2.21(4H, m), 3.14 (2H, s), 3.14 (2H, s), 3.64 (2H, t, J=6.6 Hz).

Reference Example 27 Octadeca-9,12,15-triynoic acid

To a solution of octadeca-9,12,15-triyn-1-ol (8.40 g, 32.5 mmol)obtained in Reference Example 26 and triethylamine (16.4 g, 163 mmol) indimethyl sulfoxide (97 mL), sulfur trioxide-pyridine (12.9 g, 81.3 mmol)was added, and the mixture was reacted at room temperature for 3 hours.After water treatment to terminate the reaction, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the obtained brown liquid was dissolvedin tert-butyl alcohol (130 mL) and 2-methyl-2-butene (18 mL). To thesolution, a solution of sodium dihydrogen phosphate dihydrate (11.2 g,71.5 mmol) and sodium chlorite (6.47 g, 71.5 mmol) in water (130 mL) wasadded dropwise over 5 minutes, and the mixture was then reacted at roomtemperature for 50 minutes. The reaction mixture was diluted with waterand subjected to extraction with a hexane-ethyl acetate mixed solvent,and the obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtainthe compound of interest as a yellow solid (6.13 g, 69%).

¹H-NMR (500 MHz, CDCl₃) δ: 1.12 (3H, t, J=7.6 Hz), 1.27-1.41 (6H, m),1.48 (2H, tt, J=7.1, 7.3 Hz), 1.64 (2H, tt, J=7.1, 7.6 Hz), 2.12-2.20(4H, m), 2.35 (2H, t, J=7.6 Hz), 3.14 (4H, s).

Reference Example 28 N-Methoxy-N-methyloctadeca-9,12,15-triynamide

To a solution of octadeca-9,12,15-triynoic acid (5.13 g, 18.8 mmol)obtained in Reference Example 27 and N,O-dimethylhydroxylaminehydrochloride (3.67 g, 37.7 mmol) in dichloromethane (132 mL),1-hydroxybenzimidazole hydrate (5.77 g, 37.7 mmol), triethylamine (3.81g, 37.7 mmol), and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimidehydrochloride (7.22 g, 37.7 mmol) were added, and the mixture wasreacted overnight at room temperature. After water treatment toterminate the reaction, the reaction mixture was subjected to extractionwith dichloromethane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(4.00 g, 67%).

¹H-NMR (500 MHz, CDCl₃) δ: 1.12 (3H, t, J=7.6 Hz), 1.29-1.41 (6H, m),1.48 (2H, tt, J=7.1, 7.3 Hz), 1.63 (2H, quint, J=7.3 Hz), 2.11-2.20 (4H,m), 2.41 (2H, t, J=7.3 Hz), 3.14 (4H, s), 3.18 (3H, s), 3.68 (3H, s).

Reference Example 29 Octacosa-19,22,25-triyn-11-one

To a solution of N-methoxy-N-methyloctadec-9,12,15-triynamide (1.00 g,3.17 mmol) obtained in Reference Example 28 in tetrahydrofuran (15 mL),a solution of 1 N n-decyl magnesium bromide in tetrahydrofuran (6.34 mL,6.34 mmol) was added, and the mixture was then reacted at roomtemperature for 1 hour and subsequently at 60° C. for 30 minutes. Aftertreatment with a saturated aqueous solution of ammonium chloride,volatile matter was removed under reduced pressure. The residue wassubjected to extraction with hexane, and the obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (0.40 g, 32%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.12 (3H, t, J=7.6Hz), 1.20-1.39 (20H, m), 1.47 (2H, tt, J=7.1, 7.3 Hz), 1.51-1.59 (4H,m), 2.12-2.20 (4H, m), 2.38 (4H, t, J=7.6 Hz), 3.14 (4H, s).

Reference Example 30 Octacosa-19,22,25-triyn-11-ol

To a solution of octacosa-19,22,25-triyn-11-one (0.40 g, 1.0 mmol)obtained in Reference Example 29 in methanol (3.0 mL) andtetrahydrofuran (3.0 mL), sodium borohydride (0.038 g, 1.0 mmol) wasadded, and the mixture was then reacted at room temperature for 60minutes. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.25 g, 62%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.12 (3H, t, J=7.6Hz), 1.21-1.52 (30H, m), 2.12-2.20 (4H, m), 3.14 (2H, s), 3.14 (2H, s),3.55-3.60 (1H, m).

Reference Example 313-(Dimethylamino)propyloctacosa-19,22,25-triyn-11-yl carbonate

A solution of octacosa-19,22,25-triyn-11-ol (0.25 g, 0.63 mmol) obtainedin Reference Example 30 and pyridine (0.31 g, 4.0 mmol) in toluene (6.2mL) was cooled to 0° C. in an ice bath, and a solution of triphosgene(0.13 g, 0.43 mmol) in toluene (0.9 mL) was added thereto over 2minutes. After stirring at 0° C. for 20 minutes, the reaction mixturewas heated to room temperature, stirred for 1 hour, and cooled to 0° C.again. 3-Dimethylamino-1-propanol (0.68 g, 6.6 mmol) was added thereto,and the mixture was reacted at room temperature for 2.5 hours. Aftertreatment with a saturated aqueous solution of sodium bicarbonate, thereaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (0.22 g, 66%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.1 Hz), 1.12 (3H, t, J=7.6Hz), 1.20-1.60 (30H, m), 1.84 (2H, tt, J=6.6, 7.6 Hz), 2.11-2.20 (4H,m), 2.22 (6H, s), 2.36 (2H, t, J=7.6 Hz), 3.14 (4H, s), 4.18 (2H, t,J=6.6 Hz), 4.65-4.71 (1H, m).

Example 213-(Dimethylamino)propyl(19Z,22Z,25Z)-octacosa-19,22,25-trien-11-ylcarbonate (Exemplary Compound 1-164)

To nickel(II) acetate tetrahydrate (0.104 g, 0.417 mmol), ethanol (5.0mL) was added under the hydrogen gas atmosphere, and a solution ofsodium borohydride (0.015 g, 0.417 mmol) in ethanol (2.5 mL) was added.After stirring at room temperature for 15 minutes, ethylenediamine(0.100 g, 1.67 mmol) was added, and the mixture was further stirred for15 minutes. Subsequently, a solution of3-(dimethylamino)propyloctacosa-19,22,25-triyn-11-yl carbonate (0.220 g,0.417 mmol) obtained in Reference Example 31 in ethanol (2.5 mL) wasadded thereto over 1 minute, and the mixture was reacted overnight atroom temperature under the hydrogen atmosphere. The reaction mixture wasdiluted with a hexane solution and subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid.

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (3H, t, J=6.8 Hz), 0.98 (3H, t, J=7.6Hz), 1.20-1.39 (26H, m), 1.49-1.62 (4H, m), 1.84 (2H, tt, J=6.6, 7.3Hz), 1.99-2.11 (4H, m), 2.22 (6H, s), 2.36 (2H, t, J=7.3 Hz), 2.78-2.83(4H, m), 4.18 (2H, t, J=6.6 Hz), 4.65-4.71 (1H, m), 5.28-5.43 (6H, m).

MS (ESI+) m/z 534 [M+H]⁺

HRMS (ESI+) m/z 534.4885 (−0.1 mDa).

Reference Example 32 Methyl(9Z,12R)-12-{[tert-butyl(dimethyl)silyl]oxy}octadec-9-enoate

To a solution of methyl ricinolate (16.7 g, 53.4 mmol) and imidazole(7.28 g, 107 mmol) in N,N-dimethylformamide (53.4 mL),tert-butyl(dimethyl)silane chloride (12.1 g, 80.2 mmol) was added over 2minutes, and the mixture was then reacted overnight at room temperature.After treatment with water, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(23.2 g, 99%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.03-0.06 (6H, m), 0.86-0.90 (12H, m),1.22-1.45 (18H, m), 1.62 (2H, tt, J=6.8, 7.6 Hz), 2.01 (2H, q, J=6.6Hz), 2.18 (2H, t, J=5.9 Hz), 2.30 (2H, t, J=7.6 Hz), 3.65 (1H, quint,J=5.9 Hz), 3.67 (3H, s), 5.33-5.45 (2H, m).

Reference Example 33(5R,7Z,24Z,27R)-5,27-Dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-one

To a solution of methyl(9Z,12R)-12-{[tert-butyl(dimethyl)silyl]oxy}octadec-9-enoate (12 g, 28.1mmol) obtained in Reference Example 32 in xylene (15 mL), a suspensionof sodium hydride (1.37 g, 64%, 36.6 mmol) washed in advance with hexanein xylene (5 mL) was added over 5 minute, and the mixture was thenreacted at 150° C. for 5.5 hours. The reaction mixture was cooled toroom temperature, then treated with water, and subjected to extractionwith hexane. The obtained organic layer was dried over anhydrousmagnesium sulfate, and the solvent was distilled off under reducedpressure to obtain an oil. To this oil, tetrahydrofuran (120 mL) and a 5N aqueous sodium hydroxide solution (28 mL) were added, and the mixturewas reacted at 90° C. for 5.5 hours. The reaction mixture was cooled toroom temperature, then treated with water, and subjected to extractionwith hexane. The obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (7.24 g, 67%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.03-0.06 (12H, m), 0.86-0.90 (24H, m),1.21-1.45 (36H, m), 1.51-1.59 (4H, m), 2.01 (4H, q, J=6.6 Hz), 2.18 (4H,t, J=5.9 Hz), 2.38 (4H, t, J=7.6 Hz), 3.65 (2H, quint, J=5.9 Hz),5.32-5.46 (4H, m).

Reference Example 34(5R,7Z,24Z,27R)-5,27-Dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-ol

To a solution of(5R,7Z,24Z,27R)-5,27-dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-one(5.5 g, 7.2 mmol) obtained in Reference Example 33 in methanol (22 mL)and tetrahydrofuran (22 mL), sodium borohydride (0.27 g, 7.2 mmol) wasadded over 2 minutes, and the mixture was then reacted at roomtemperature for 3 hours. After treatment with a saturated aqueoussolution of ammonium chloride, volatile matter was removed under reducedpressure. The residue was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (5.0 g, 91%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.03-0.05 (12H, m), 0.86-0.90 (24H, m),1.22-1.47 (44H, m), 2.01 (4H, q, J=6.6 Hz), 2.18 (4H, t, J=5.9 Hz),3.55-3.61 (1H, m), 3.65 (2H, quint, J=5.9 Hz), 5.34-5.46 (4H, m).

Reference Example 353-(Dimethylamino)propyl(5R,7Z,24Z,27R)-5,27-dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-ylcarbonate

To a solution of(5R,7Z,24Z,27R)-5,27-dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-ol(1.00 g, 1.31 mmol) obtained in Reference Example 34 and pyridine (0.651g, 8.23 mmol) in toluene (13.1 mL), a solution of triphosgene (0.268 g,0.690 mmol) in toluene (1.96 mL) was added over 1 minute. After stirringat room temperature for 2 hours, 3-dimethylamino-1-propanol (1.42 g,13.7 mmol) was added thereto, and the mixture was reacted at roomtemperature for 3 hours. After treatment with a saturated aqueoussolution of sodium bicarbonate, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(1.15 g, 98%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.03-0.05 (12H, m), 0.88 (6H, t, J=6.8 Hz),0.89 (18H, s), 1.21-1.46 (40H, m), 1.49-1.62 (4H, m), 1.84 (2H, tt,J=6.8, 7.3 Hz), 2.01 (4H, q, J=6.6 Hz), 2.18 (4H, t, J=5.9 Hz), 2.22(2H, t, J=7.3 Hz), 3.65 (2H, quint, J=5.9 Hz), 4.17 (2H, t, J=6.8 Hz),4.65-4.71 (1H, m), 5.33-5.46 (4H, m).

Example 223-(Dimethylamino)propyl(7R,9Z,26Z,29R)-7,29-dihydroxypentatriaconta-9,26-dien-18-ylcarbonate (Exemplary Compound 1-308)

To3-(dimethylamino)propyl(5R,7Z,24Z,27R)-5,27-dihexyl-2,2,3,3,29,29,30,30-octamethyl-4,28-dioxa-3,29-disilahentriaconta-7,24-dien-16-ylcarbonate (1.15 g, 1.29 mmol) obtained in Reference Example 35, asolution of 1 N tetra-n-butylammonium fluoride in tetrahydrofuran (19.3mL, 19.3 mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.56 g, 65%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (6H, t, J=6.8 Hz), 1.23-1.38 (36H, m),1.40-1.50 (4H, m), 1.50-1.62 (4H, m), 1.84 (2H, tt, J=6.8, 7.3 Hz), 2.04(4H, q, J=7.1 Hz), 2.21 (4H, t, J=7.1 Hz), 2.22 (6H, s), 2.36 (2H, t,J=7.3 Hz), 3.57-3.64 (2H, m), 4.17 (2H, t, J=6.8 Hz), 4.65-4.71 (1H, m),5.37-5.44 (1H, m), 5.54-5.58 (1H, m).

Example 23(7R,9Z,26Z,29R)-18-({[3-(Dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyldiacetate (Exemplary Compound 1-233)

To a solution of3-(dimethylamino)propyl(7R,9Z,26Z,29R)-7,29-dihydroxypentatriaconta-9,26-dien-18-ylcarbonate (0.15 g, 0.23 mmol) obtained in Example 22 and pyridine (0.36g, 4.5 mmol) in dichloromethane (4.5 mL), acetic acid chloride (0.18 g,2.3 mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withdichloromethane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (155 mg, 92%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.38 (40H, m),1.49-1.63 (8H, m), 1.85 (2H, tt, J=6.6, 7.3 Hz), 1.98-2.05 (10H, m),2.22 (6H, s), 2.24-2.33 (4H, m), 2.36 (2H, t, J=7.3 Hz), 4.18 (2H, t,J=6.6 Hz), 4.65-4.71 (1H, m), 4.87 (2H, quint, J=6.3 Hz), 5.29-5.36 (2H,m), 5.44-5.50 (2H, m).

MS (ESI+) m/z 750 [M+H]⁺

HRMS (ESI+) m/z 750.6247 (−0.1 mDa).

Example 24(7R,9Z,26Z,29R)-7,29-Dihexyl-2,5-dioxo-1,6-dioxacyclononacosa-9,26-dien-18-yl3-(dimethylamino)propyl carbonate (Exemplary Compound 1-319)

To a solution of3-(dimethylamino)propyl(7R,9Z,26Z,29R)-7,29-dihydroxypentatriaconta-9,26-dien-18-ylcarbonate (1.19 g, 1.72 mmol) obtained in Example 22 and pyridine (1.13g, 14.3 mmol) in dichloromethane (40 mL), a solution of succinic acidchloride (0.186 g, 1.20 mmol) in dichloromethane (13 mL) was addeddropwise over 1.5 hours, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withdichloromethane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (20 mg, 1%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.88 (6H, t, J=6.6 Hz), 1.21-1.39 (40H, m),1.51-1.63 (8H, m), 1.84 (2H, tt, J=6.6, 7.3 Hz), 1.98-2.07 (4H, m), 2.22(6H, s), 2.28 (2H, t, J=7.1 Hz), 2.32 (2H, t, J=7.3 Hz), 2.36 (2H, t,J=7.6 Hz), 2.60 (4H, s), 4.17 (2H, t, J=6.6 Hz), 4.68 (1H, quint, J=6.1Hz), 4.86-4.92 (2H, m), 5.29-5.36 (2H, m), 5.43-5.50 (2H, m).

MS (ESI+) m/z 748 [M+H]⁺

HRMS (ESI+) m/z 748.6091 (0.0 mDa).

Reference Example 36 (11Z,14Z)-N-Methoxy-N-methylicosa-11,14-dienamide

To a solution of (11Z,14Z)-icosa-11,14-dienoate (compound 3 described inChem. Lett. 1998, 2, 175, 4.45 g, 14.4 mmol) andN,O-dimethylhydroxylamine hydrochloride (2.87 g, 28.9 mmol) indichloromethane (71 mL), 1-hydroxybenzimidazole hydrate (3.90 g, 28.9mmol), triethylamine (2.95 g, 28.9 mmol), and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (5.53 g,28.9 mmol) were added, and the mixture was reacted overnight at roomtemperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (5.00 g, 99%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 1.24-1.40 (18H, m),1.62 (2H, quint, J=7.4 Hz), 2.02-2.07 (4H, m), 2.41 (2H, t, J=7.4 Hz),2.77 (2H, t, J=6.6 Hz), 3.18 (3H, s), 3.68 (3H, s), 5.29-5.43 (4H, m).

Reference Example 37 (21Z,24Z)-Triaconta-21,24-dien-11-ol

To a solution of (11Z,14Z)-N-methoxy-N-methylicosa-11,14-dienamide (0.50g, 1.4 mmol) obtained in Reference Example 36 in tetrahydrofuran (6.3mL), a solution of 1 N n-decyl magnesium bromide in diethyl ether (4.3mL, 4.3 mmol) was added dropwise over 3 minutes, and the mixture wasthen reacted at room temperature for 1.5 hours. After treatment with asaturated aqueous solution of ammonium chloride, the reaction mixturewas subjected to extraction with ethyl acetate, and the obtained organiclayer was dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the residue was then subjectedto silica gel column chromatography. To a solution of the obtained oilin methanol (5.4 mL) and tetrahydrofuran (5.4 mL), sodium borohydride(0.05 g, 1.3 mmol) was added, and the mixture was then reacted at roomtemperature for 30 minutes. After treatment with a saturated aqueoussolution of ammonium chloride, the reaction mixture was subjected toextraction with ethyl acetate, and the obtained organic layer was driedover anhydrous magnesium sulfate. The solvent was distilled off underreduced pressure to obtain a mixture containing the compound ofinterest.

Example 25 3-(Dimethylamino)propyl(21Z,24Z)-triaconta-21,24-dien-11-ylcarbonate (Exemplary Compound 1-444)

To a solution of (21Z,24Z)-triaconta-21,24-dien-11-ol (0.15 g, 0.35mmol) obtained in Reference Example 37 and pyridine (0.17 g, 2.18 mmol)in toluene (0.7 mL), a solution of triphosgene (0.07 g, 0.25 mmol) intoluene (0.29 mL) was added over 2 minutes. After stirring at roomtemperature for 2 hours, 3-dimethylamino 1-propanol (0.37 g, 3.6 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (100 mg, 51%).

¹H-NMR (500 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.21-1.40 (36H, m),1.49-1.61 (4H, m), 1.85 (2H, tt, J=6.6, 7.6 Hz), 2.01-2.08 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.6 Hz), 2.77 (2H, t, J=6.6 Hz), 4.18 (2H, t,J=6.6 Hz), 4.65-4.72 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 564 [M+H]⁺

HRMS (ESI+) m/z 564.5352 (−0.4 mDa).

Example 26 (19Z,22Z)-Octacosa-19,22-dien-11-yl 3-(pyrrolidin-1-yl)propylcarbonate (Exemplary Compound 1-77)

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.200 g, 0.492 mmol)obtained in Reference Example 17 and pyridine (0.245 g, 3.10 mmol) intoluene (4.9 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.101 g, 0.339 mmol) in toluene (0.74 mL) was added theretoover 1 minute. After stirring at 0° C. for 15 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. 3-(Pyrrolidin-1-yl)propan-1-ol (0.667 g, 5.16 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (127 mg, 46%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=6.7 Hz), 0.89 (3H, t, J=6.7Hz), 1.20-1.40 (32H, m), 1.48-1.64 (4H, m), 1.74-1.81 (4H, m), 1.90 (2H,tt, J=6.7, 7.4 Hz), 2.01-2.09 (4H, m), 2.45-2.54 (4H, m), 2.53 (2H, t,J=7.4 Hz), 2.77 (2H, t, J=6.7 Hz), 4.19 (2H, t, J=6.7 Hz), 4.70 (1H, tt,J=5.5, 7.0 Hz), 5.28-5.43 (4H, m).

MS (ESI+) m/z 562 [M+H]⁺

HRMS (ESI+) m/z 562.5203 (0.4 mDa).

Reference Example 38(19Z,22R)-22-{[tert-Butyl(dimethyl)silyl]oxy}octacos-19-en-11-one

To a solution of methyl(9Z,12R)-12-{[tert-butyl(dimethyl)silyl]oxy}octadec-9-enoate (2.00 g,4.69 mmol) obtained in Reference Example 32 and methyl undecanoate (2.82g, 14.1 mmol) in xylene (20 mL), a suspension of sodium hydride (0.879g, 64%, 23.4 mmol) washed in advance with hexane in xylene (8 mL) wasadded over 5 minutes, and the mixture was then reacted at 150° C. for6.5 hours. The reaction mixture was cooled to room temperature, thentreated with water, and subjected to extraction with hexane-ethylacetate. The obtained organic layer was dried over anhydrous magnesiumsulfate, and the solvent was distilled off under reduced pressure toobtain an oil. To this oil, tetrahydrofuran (94 mL) and a 5 N aqueoussodium hydroxide solution (23 mL) were added, and the mixture wasreacted at 90° C. for 5 hours. The reaction mixture was cooled to roomtemperature, then treated with water, and subjected to extraction withhexane. The obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtaina liquid containing the compound of interest (3.41 g, containingimpurities).

¹H-NMR (400 MHz, CDCl₃) δ: 0.03-0.05 (6H, m), 0.85-0.91 (15H, m),1.21-1.44 (36H, m), 1.50-1.60 (4H, m), 1.97-2.04 (2H, m), 2.18 (2H, t,J=6.3 Hz), 2.38 (4H, t, J=7.4 Hz), 3.61-3.68 (1H, m), 5.33-5.46 (2H, m).

Reference Example 39(19Z,22R)-22-{[tert-Butyl(dimethyl)silyl]oxy}octacos-19-en-11-ol

To a solution of the mixture containing(19Z,22R)-22-{[tert-butyl(dimethyl)silyl]oxy}octacos-19-en-11-one (3.41g)) obtained in Reference Example 38 in methanol (24 mL) andtetrahydrofuran (24 mL), sodium borohydride (0.30 g, 8.0 mmol) was addedover 2 minutes, and the mixture was then reacted at room temperature for1.5 hours. After treatment with a saturated aqueous solution of ammoniumchloride, volatile matter was removed under reduced pressure. Theresidue was subjected to extraction with hexane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain a liquidcontaining the compound of interest (3.54 g, containing impurities).

Reference Example 40(19Z,22R)-22-{[tert-Butyl(dimethyl)silyl]oxy}octacos-19-en-11-yl3-(dimethylamino)propyl carbonate

A solution of the mixture containing(19Z,22R)-22-{[tert-butyl(dimethyl)silyl]oxy}octacos-19-en-11-ol (3.54g) obtained in Reference Example 39 and pyridine (4.00 g, 50.6 mmol) intoluene (78.8 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (1.62 g, 5.45 mmol) in toluene (11.8 mL) was added theretoover 2 minutes. After stirring at 0° C. for 30 minutes, the reactionmixture was heated to room temperature, stirred for 1 hour, and cooledto 0° C. again. 3-Dimethylamino-1-propanol (8.81 g, 85.4 mmol) was addedthereto, and the mixture was reacted overnight at room temperature.After treatment with a saturated aqueous solution of sodium bicarbonate,the reaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain a liquidcontaining the compound of interest (2.58 g, containing impurities).

¹H-NMR (400 MHz, CDCl₃) δ: 0.03-0.06 (6H, m), 0.85-0.91 (15H, m),1.23-1.64 (40H, m), 1.84 (2H, tt, J=6.7, 7.4 Hz), 1.97-2.04 (2H, m),2.15-2.20 (2H, m), 2.22 (6H, s), 2.36 (2H, t, J=7.4 Hz), 3.61-3.68 (1H,m), 4.17 (2H, t, J=6.7 Hz), 4.69 (1H, tt, J=5.5, 7.0 Hz), 5.32-5.46 (2H,m).

Example 273-(Dimethylamino)propyl(19Z,22R)-22-hydroxyoctacos-19-en-11-yl carbonate(Exemplary Compound 1-298)

To the mixture containing(19Z,22R)-22-{[tert-butyl(dimethyl)silyl]oxy}octacos-19-en-11-yl3-(dimethylamino)propyl carbonate (2.58 g,) obtained in ReferenceExample 40, a solution of 1 N tetra-n-butylammonium fluoride intetrahydrofuran (23.2 mL, 23.2 mmol) was added, and the mixture wasreacted overnight at room temperature. After water treatment toterminate the reaction, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.500 g).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=6.7 Hz), 0.89 (3H, t, J=6.7Hz), 1.20-1.38 (28H, m), 1.40-1.62 (8H, m), 1.84 (2H, tt, J=6.7, 7.4Hz), 2.01-2.08 (2H, m), 2.18-2.23 (2H, m), 2.22 (6H, s), 2.36 (2H, t,J=7.4 Hz), 3.61 (1H, tt, J=5.3, 5.9 Hz), 4.18 (2H, t, J=6.7 Hz), 4.69(1H, tt, J=5.5, 6.3 Hz), 5.36-5.45 (1H, m), 5.52-5.60 (1H, m).

MS (ESI+) m/z 554 [M+H]⁺

HRMS (ESI+) m/z 554.5146 (−0.2 mDa).

Example 28(7R,9Z)-18-({[3-(Dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-ylacetate (Exemplary Compound 1-212)

To a solution of3-(dimethylamino)propyl(19Z,22R)-22-hydroxyoctacos-19-en-11-yl carbonate(0.20 g, 0.36 mmol) obtained in Example 27 and pyridine (0.57 g, 7.2mmol) in dichloromethane (7.2 mL), acetyl chloride (0.28 g, 3.6 mmol)was added, and the mixture was reacted at room temperature for 3 hours.After treatment with a saturated aqueous solution of sodium bicarbonate,the reaction mixture was subjected to extraction with dichloromethane,and the obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtainthe compound of interest as a colorless liquid (42 mg, 20%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=6.7 Hz), 0.89 (3H, t, J=6.7Hz), 1.20-1.37 (32H, m), 1.48-1.61 (4H, m), 1.85 (2H, tt, J=6.7, 7.4Hz), 1.97-2.02 (2H, m), 2.03 (3H, s), 2.22 (6H, s), 2.28 (2H, dd, J=6.3,7.0 Hz), 2.36 (2H, t, J=7.4 Hz), 4.18 (2H, t, J=6.7 Hz), 4.69 (1H, tt,J=5.5, 6.3 Hz), 4.87 (1H, quint, J=6.3 Hz), 5.32 (1H, dtt, J=11.0, 1.6,7.0 Hz), 5.47 (dtt, J=11.0, 1.6, 7.0 Hz).

MS (ESI+) m/z 596 [M+H]⁺

HRMS (ESI+) m/z 596.5269 (1.5 mDa).

Reference Example 411,15-Bis[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]pentadecan-8-one

To a solution of methyl8-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octanoate(intermediate of compound 6 described in Bioorg. Med. Chem. Lett. 2003,13, 1037, 1.50 g, 4.48 mmol) in xylene (6.0 mL), a suspension of sodiumhydride (0.219 g, 64%, 5.83 mmol) washed in advance with hexane inxylene (0.7 mL) was added over 2 minutes, and the mixture was thenreacted at 150° C. for 5 hours. The reaction mixture was cooled to roomtemperature, then treated with water, and subjected to extraction withhexane. The obtained organic layer was dried over anhydrous magnesiumsulfate, and the solvent was distilled off under reduced pressure toobtain an oil. To this oil, tetrahydrofuran (22.4 mL) and a 5 N aqueoussodium hydroxide solution (5.4 mL) were added, and the mixture wasreacted at 90° C. for 5 hours. The reaction mixture was cooled to roomtemperature, then treated with water, and subjected to extraction with ahexane-ethyl acetate mixed solution. The obtained organic layer wasdried over anhydrous magnesium sulfate. The solvent was distilled offunder reduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (0.294 g, 23%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.17) (6H, m), 0.57-1.61 (60H, m),2.38 (4H, t, J=7.4 Hz).

Reference Example 421,15-Bis[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]pentadecan-8-ol

To a solution of1,15-bis[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]pentadecan-8-one(0.29 g, 0.51 mmol) obtained in Reference Example 41 in methanol (1.5mL) and tetrahydrofuran (1.5 mL), sodium borohydride (0.019 g, 0.51mmol) was added, and the mixture was then reacted at room temperaturefor 90 minutes. After treatment with a saturated aqueous solution ofammonium chloride, the reaction mixture was subjected to extraction withethyl acetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.23 g, 78%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.16) (6H, m), 0.57-1.60 (64H, m),3.54-3.63 (1H, m).

Example 291,15-Bis[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]pentadecan-8-yl3-dimethylaminopropyl carbonate (Exemplary Compound 1-200)

A solution of1,15-bis[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]pentadecan-8-ol(0.13 g, 0.22 mmol) obtained in Reference Example 42 and pyridine (0.11g, 1.4 mmol) in toluene (2.2 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.046 g, 0.15 mmol) in toluene (0.34 mL) wasadded thereto over 2 minutes. The resulting solution was stirred at 0°C. for 20 minutes, then heated to room temperature, stirred for 40minutes, and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.24 g,2.4 mmol) was added thereto, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (150 mg, 94%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.16) (6H, m), 0.56-1.66 (64H, m),1.84 (2H, tt, J=6.7, 7.4 Hz), 2.22 (6H, s), 2.36 (2H, t, J=7.4 Hz), 4.17(2H, t, J=6.7 Hz), 4.70 (1H, tt, J=5.5, 7.0 Hz).

MS (ESI+) m/z 710 [M+H]⁺

HRMS (ESI+) m/z 710.6463 (1.2 mDa).

Reference Example 438-[2-({2-[(2-Ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]-N-methoxy-N-methyloctanamide

To a solution of methyl8-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octanoate(compound 6 described in Bioorg. Med. Chem. Lett. 2003, 13, 1037, 3.00g, 9.36 mmol) and N,O-dimethylhydroxylamine hydrochloride (1.83 g, 18.7mmol) in dichloromethane (65.5 mL), 1-hydroxybenzimidazole hydrate (2.87g, 18.7 mmol), triethylamine (1.89 g, 18.7 mmol), and1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (3.59 g,18.7 mmol) were added, and the mixture was reacted overnight at roomtemperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (3.08 g, 91%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.17) (3H, m), 0.57-1.56 (28H, m),1.57-1.67 (2H, m), 2.41 (2H, t, J=7.4 Hz), 3.18 (3H, s), 3.68 (3H, s).

Reference Example 441-[2-({2-[(2-Ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octadecan-8-one

A solution of8-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]-N-methoxy-N-methyloctanamide(2.00 g, 5.50 mmol) obtained in Reference Example 43 in tetrahydrofuran(24.5 mL) was cooled to 15° C. in a water bath. A solution of 1 Nn-decyl magnesium bromide in tetrahydrofuran (8.25 mL, 8.25 mmol) wasadded dropwise thereto over 15 minutes, and the mixture was then reactedovernight at room temperature. After treatment with a saturated aqueoussolution of ammonium chloride, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(1.56 g, 64%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.17) (3H, m), 0.57-1.62 (49H, m),2.38 (4H, t, J=7.4 Hz).

Reference Example 451-[2-({2-[(2-Ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octadecan-8-ol

To a solution of1-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octadecan-8-one(1.56 g, 3.51 mmol) obtained in Reference Example 44 in methanol (10.5mL) and tetrahydrofuran (10.5 mL), sodium borohydride (0.133 g, 3.51mmol) was added, and the mixture was then reacted at room temperaturefor 90 minutes. After treatment with a saturated aqueous solution ofammonium chloride, the reaction mixture was subjected to extraction withethyl acetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (1.50 g, 96%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.17) (3H, m), 0.57-1.56 (53H, m),3.54-3.62 (1H, m).

Example 303-Dimethylaminopropyl1-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octadecan-8-ylcarbonate (Exemplary Compound 1-188)

A solution of1-[2-({2-[(2-ethylcyclopropyl)methyl]cyclopropyl}methyl)cyclopropyl]octadecan-8-ol(0.25 g, 0.56 mmol) obtained in Reference Example 45 and pyridine (0.28g, 3.5 mmol) in toluene (5.6 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.12 g, 0.39 mmol) in toluene (0.84 mL) wasadded thereto over 2 minutes. The resulting solution was stirred at 0°C. for 20 minutes, then heated to room temperature, stirred for 40minutes, and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.61 g,5.9 mmol) was added thereto, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (155 mg, 48%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.16) (3H, m), 0.57-1.65 (53H, m),1.84 (2H, tt, J=6.7, 7.4 Hz), 2.22 (6H, s), 2.36 (2H, t, J=7.4 Hz), 4.18(2H, t, J=6.7 Hz), 4.67 (1H, tt, J=5.5, 7.0 Hz).

MS (ESI+) m/z 576 [M+H]⁺

HRMS (ESI+) m/z 576.5367 (1.1 mDa).

Example 31 Preparation of Double-stranded Polynucleotide-encapsulatedNucleic Acid Lipid Particle

A lipid solution having a total lipid concentration of 25 mM in 90%ethanol with distearoylphosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter, referred to asDSPC, NOF CORPORATION), cholesterol (hereinafter, referred to as Chol,Sigma-Aldrich, Inc.), the compound described in Example 1, 2, 3, or 8(hereinafter, referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]4,2-dimyristyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DMA) were prepared at a molar ratio ofDSPC:Chol:LP:PEG-C-DMA=20:48:30:2.

A polynucleotide CT-157:

HO—P(═O)(OH)—O-U^(mlp)-T^(p)-G^(mlp)-T^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-C^(mlp)-A^(p)-U^(mlp)-T^(p)-C^(mlp)-T^(p)-U^(mlp)-G^(p)-U^(mlp)-G^(p)-C^(mlp)-T^(p)-U^(mlt)-H(SEQ ID NO: 2 of the Sequence Listing) (polynucleotide containing

a sequence complementary to nucleotide positions 3139-3157 of the humanβ-catenin gene (GenBank accession No. NM_001904.3)) and

a polynucleotide CT-169:

HO-G^(p)-C^(mlp)-A^(p)-C^(mlp)-A^(p)-A^(mlp)-G^(p)-A^(mlp)-A^(p)-U^(mlp)-G^(p)-G^(mlp)-A^(p)-U^(mlp)-C^(p)-A^(mlp)-C^(p)-A^(mlt)-H(SEQ ID NO: 1 of the Sequence Listing) (containing a sequence ofnucleotide positions 3139-3156 of the human β-catenin gene (GenBankaccession No. NM_001904.3)) described in Examples 45 and 51 ofInternational Publication No. WO 2010/001909 were synthesized using aDNA synthesizer, placed in an amount of 300 pmol/tube, and dried underreduced pressure. 30 μL of an siRNA suspension buffer (Qiagen N.V.) wasadded thereto, and the mixture was heated at 65° C. for 1 minute andthen left at room temperature for 5 minutes for annealing to obtain a 10μM double-stranded polynucleotide solution. Then, the concentration ofthe solution was adjusted to 1 mg/mL with a citrate buffer solution (20mM citrate buffer, pH 4.0) to obtain a double-stranded polynucleotidesolution. The lipid solution and the double-stranded polynucleotidesolution were heated to 37° C. and mixed (100 μL each). Subsequently,200 μL of a citrate buffer solution (20 mM citrate buffer, 300 mM NaCl,pH 6.0) was added thereto, and the mixture was incubated at 37° C. for30 minutes to obtain a nucleic acid lipid particle dispersion. Thenucleic acid lipid particle dispersion was dialyzed againstapproximately 100 mL of a phosphate buffer solution (pH 7.4) for 12 to18 hours (Float-A-Lyzer G2, MWCO: 100 kD, Spectra/Por) for the removalof ethanol and the removal of unencapsulated double-strandedpolynucleotides by neutralization to obtain a purified dispersion of ansiRNA-encapsulated nucleic acid lipid particle containing the compounddescribed in Example 1, 2, 3, or 8. The control samples used were thecompound described in Reference Example 2 and compound 1 described inWO2012/054365.

Example 32 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

The nucleic acid lipid particle-containing dispersion prepared inExample 31 was characterized. Each characterization method will bedescribed.

(1) Average Particle Size

The particle size of the liposome was measured using ZetaPotential/Particle Sizer NICOMP™ 380ZLS (Particle Sizing Systems, LLC).In the tables, the average particle size is indicated by avolume-average particle size, and the numeric value following±representsa deviation.

(2) Rate of Encapsulation of Double-stranded Polynucleotide

The rate of encapsulation of the double-stranded polynucleotide wasmeasured using Quant-iT RiboGreen RNA Assay kit (Invitrogen Corp.)according to the attached document.

Specifically, the double-stranded polynucleotide in the nucleic acidlipid particle dispersion was quantified in the presence and absence ofa 0.015% Triton X-100 detergent, and the rate of encapsulation wascalculated according to the following expression:{[Amount of the double-stranded polynucleotide in the presence of thedetergent]−[Amount of the double-stranded polynucleotide in the absenceof the detergent]}/[Amount of the double-stranded polynucleotide in thepresence of the detergent]}×100 (%)(3) Ratio of Double-stranded Polynucleotide to Lipid

The nucleic acid lipid particle dispersion was mixed with acetonitrileand chloroform at a ratio of 1:1:1 and centrifuged at 15,000 rpm for 2minutes. Then, the aqueous layer obtained as an upper layer wasrecovered, followed by the extraction of the double-strandedpolynucleotide. The amount of the double-stranded polynucleotide in thesample was measured by ion-exchange chromatography (system: Agilent 1100series, column: TSKgel DEAE-2SW (2.6×150 mm) (Tosoh Corp), buffer A: 20%acetonitrile, buffer B: 20% acetonitrile and 1.6 M ammonium formate,gradient (B%): 30-55% (0-20 min), flow rate: 1 mL/min, temperature: 40°C., detection: 260 nm).

The amount of the phospholipid in the nucleic acid lipid particledispersion was measured using Phospholipid C-Test Wako (Wako PureChemical Industries Ltd.) according to the attached document.Specifically, the phospholipid in the sample was quantified in thepresence of a 1% Triton X-100 detergent.

The amounts of cholesterol and LP in the nucleic acid lipid particledispersion were measured by reverse-phase chromatography (system:Agilent 1100 series, column: Chromolith Performance RP-18 endcapped100-3 monolithic HPLC-column (Merck), buffer A: 0.01% trifluoroaceticacid, buffer B: 0.01% trifluoroacetic acid and methanol, gradient (B%):87-92% (0-10 min), flow rate: 2 mL/min, temperature: 50° C., detection:205 nm).

The total amount of lipids was calculated from the amount of thephospholipid and the compositional ratio of lipid componentsconstituting the liposome, and the ratio of the polynucleotide to thelipid was calculated from the aforementioned amount of thepolynucleotide and the total amount of lipids according to the followingexpression:[Double-stranded polynucleotide concentration]/[Total lipidconcentration] (wt/wt)

The results are shown in Table 3.

TABLE 3 Ratio of Rate of polynucleotide polynucleotide to lipid Averageencapsulation siRNA/lipid particle size LP name (%) (wt/wt) (nm)Reference Example 2 89.0 0.104 139 ± 50 Example 1 94.3 0.109 183 ± 41Example 2 96.3 0.099 215 ± 70 Example 3 98.6 0.099 187 ± 31

The total amount of lipids was calculated from the amount of thephospholipid, the amount of cholesterol, and the amount of LP, and thecompositional ratio of lipid components constituting the liposome, andthe ratio of the polynucleotide to the lipid was calculated from theaforementioned amount of the polynucleotide and the total amount oflipids according to the following expression:[Double-stranded polynucleotide concentration]/[Total lipidconcentration] (wt/wt)

The results are shown in Table 4.

TABLE 4 Ratio of Rate of polynucleotide polynucleotide to lipid Averageencapsulation siRNA/lipid particle size LP name (%) (wt/wt) (nm)Compound 1 93.0 0.075 138 ± 24 Example 8 96.4 0.072 157 ± 46

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 100 nm to approximately300 nm.

Test Example 1

As described below, the strength of human β-catenin gene expressioninhibitory activity was compared among nucleic acid lipid particles eachprepared using a novel lipid.

(1) Transfection

The concentration of a human colorectal cancer SW480 cell line (derivedfrom human colorectal adenocarcinoma) was adjusted to 50,000 cells/mL inan RPMI1640 medium (manufactured by Invitrogen Corp.) containing 10%fetal bovine serum (culture medium). Then, the resulting culturesolution was inoculated at 100 μL/well to a 96-well flat-bottomed plate(manufactured by Corning Inc./Falcon) and cultured at 37° C. for 1 dayunder 5.0% CO₂. The nucleic acid lipid particle dispersion prepared inExample 31 was diluted with a culture medium to prepare dilution serieshaving final double-stranded polynucleotide concentrations of 30, 3.0,0.3, and 0.03 nM in the medium. Then, each dilution was added to thecells after removal of the culture supernatant, and the culture wasfurther continued for 3 days. This operation was performed at N=3 foreach concentration.

(2) Real-time PCR

A lysate and cDNA for real-time PCR measurement were prepared from thetransfected cells using TaqMan(R) Fast-Cells-to-Ct kit (LifeTechnologies, Inc./Ambion) according to the instruction manual. In thelysate preparation, Lysis Solution supplemented with DNase I was used.The probes for real-time PCR used were TaqMan(R) Gene Expression Assays(CTNNB1, FAM probe) (Hs00355045_m1, manufactured by Applied Biosystems,Inc.) for the human β-catenin gene and a human GAPDH gene probe as aninternal standard (VIC probe, Hs99999905_m1, manufactured by AppliedBiosystems, Inc.). 5 μL of TaqMan(R) Fast Advanced Master Mix, 2 μL ofRNase-Free Water, 0.5 μL of each gene probe, and 2 μL of the preparedcDNA solution were added per well of a 384-well PCR plate (manufacturedby Applied Biosystems, Inc.) to bring the total amount to 10 μL, whichwas then loaded in ViiA™ 7 Real-time PCR system (manufactured by AppliedBiosystems, Inc.) and subjected to PCR under conditions given below. Thereal-time PCR was carried out at N=4 for the cDNA prepared from thelysate.

PCR initial activation: 95° C. for 20 seconds

PCR: 95° C. for 1 second

-   -   62° C. for 20 seconds

This PCR cycle was repetitively performed 40 times.

(3) Real-time PCR Analysis

The quantitative analysis was conducted by the ΔΔCt method. A value(ΔΔCt) was determined by subtracting ΔCt of an untreated cell (═NC) fromthe difference in Ct value (ΔCt) between human β-catenin and human GAPDHof each transfectant, and a relative value (RQ) to NC was calculatedaccording to the following expression:RQ=2^(−ΔΔCt)When RQ=1 was defined as 0% rate of inhibition and RQ=0 was defined astheoretical 100% rate of inhibition, the IC₅₀ value of the nucleic acidlipid particle was calculated using GraphPad PRISM (GraphPad SoftwareInc.). As a result, as shown in Table 5, the nucleic acid lipid particlecontaining the compound of Example 1, 2, or 3 exhibited stronginhibitory activity against β-catenin gene expression, as compared withthe nucleic acid lipid particle containing the lipid of ReferenceExample 2 used as a control. These results demonstrated that thecompounds of Examples 1, 2, and 3 are novel lipids useful for preparingnucleic acid lipid particles that exhibit strong activity.

TABLE 5 β-catenin gene expression inhibitory activity IC50 (nM)Reference Example 2 >30 Example 1 3.5 Example 2 6.8 Example 3 0.44

Test Example 2

As described below, the strength of human β-catenin gene expressioninhibitory activity was compared among nucleic acid lipid particles eachprepared using a novel lipid.

(1) Transfection

The concentration of a human liver cancer HepG2 cell line (derived fromhuman liver cancer) was adjusted to 50000 cells/mL in a DMEM medium(manufactured by Invitrogen Corp.) containing 10% fetal bovine serum(culture medium). Then, the resulting culture solution was inoculated at100 μL/well to a 96-well flat-bottomed plate (manufactured by CorningInc./Falcon) and cultured at 37° C. for 1 day under 5.0% CO₂. Thenucleic acid lipid particle-containing dispersion prepared in Example 31was diluted with a culture medium to prepare dilution series havingfinal double-stranded polynucleotide concentrations of 30, 3, 0.3, and0.03 nM in the medium. Then, each dilution was added to the cells afterremoval of the culture supernatant, and the culture was furthercontinued for 3 days. This operation was performed at N=3 for eachconcentration.

(2) Real-time PCR

A lysate and cDNA for real-time PCR measurement were prepared from thetransfected cells using TaqMan(R) Fast-Cells-to-Ct kit (LifeTechnologies, Inc./Ambion) according to the instruction manual. In thelysate preparation, Lysis Solution supplemented with DNase I was used.The probes for real-time PCR used were TaqMan(R) Gene Expression Assays(CTNNB1, FAM probe) (Hs00355045_m1, manufactured by Applied Biosystems,Inc.) for the human β-catenin gene and a human GAPDH gene probe as aninternal standard (VIC probe, Hs99999905_m1, manufactured by AppliedBiosystems, Inc.). 5 μL of TaqMan(R) Fast Advanced Master Mix, 2 μL ofRNase-Free Water, 0.5 μL of each gene probe, and 2 μL of the preparedcDNA solution were added per well of a 384-well PCR plate (manufacturedby Applied Biosystems, Inc.) to bring the total amount to 10 μL, whichwas then loaded in ViiA™ 7 Real-time PCR system (manufactured by AppliedBiosystems, Inc.) and subjected to PCR under conditions given below. Thereal-time PCR was carried out at N=4 for the cDNA prepared from thelysate.

PCR initial activation: 95° C. for 20 seconds

PCR: 95° C. for 1 second

-   -   62° C. for 20 seconds

This PCR cycle was repetitively performed 40 times.

(3) Real-time PCR Analysis

The quantitative analysis was conducted by the ΔΔCt method. A value(ΔΔCt) was determined by subtracting ΔCt of an untreated cell (═NC) fromthe difference in Ct value (ΔCt) between human β-catenin and human GAPDHof each transfectant, and a relative value (RQ) to NC was calculatedaccording to the following expression:RQ=2^(−ΔΔCt)When RQ=1 was defined as 0% rate of inhibition and RQ=0 was defined astheoretical 100% rate of inhibition, the IC₅₀ value of the nucleic acidlipid particle was calculated using GraphPad PRISM (GraphPad SoftwareInc.). As a result, as shown in Table 6, the nucleic acid lipid particlecontaining the compound of Example 1, 2, or 3 exhibited stronginhibitory activity against β-catenin gene expression, as compared withthe nucleic acid lipid particle containing the lipid of ReferenceExample 2 used as a control. These results demonstrated that thecompounds of Examples 1, 2, and 3 are novel lipids useful for preparingnucleic acid lipid particles that exhibit strong activity.

TABLE 6 β-catenin gene expression inhibitory activity IC50 (nM)Reference Example 2 >30 Example 1 0.38 Example 2 0.47 Example 3 0.35

Test Example 3

As described below, the strength of human β-catenin gene expressioninhibitory activity was compared among nucleic acid lipid particles eachprepared using a novel lipid.

(1) Transfection

The concentration of a human colorectal cancer SW480 cell line (derivedfrom human colorectal adenocarcinoma) was adjusted to 50000 cells/mL ina RPMI1640 medium (manufactured by Invitrogen Corp.) containing 10%fetal bovine serum (culture medium). Then, the resulting culturesolution was inoculated at 100 μL/well to a 96-well flat-bottomed plate(manufactured by Corning Inc./Falcon) and cultured at 37° C. for 1 dayunder 5.0% CO₂. The nucleic acid lipid particle-containing dispersionprepared in Example 31 was diluted with a culture medium to preparedilution series having final double-stranded polynucleotideconcentrations of 30, 3, 0.3, and 0.03 nM in the medium. Then, eachdilution was added to the cells after removal of the culturesupernatant, and the culture was further continued for 4 hours. Thisoperation was performed at N=3 for each concentration.

(2) Real-time PCR

A lysate and cDNA for real-time PCR measurement were prepared from thetransfected cells using TaqMan(R) Fast-Cells-to-Ct kit (LifeTechnologies, Inc./Ambion) according to the instruction manual. In thelysate preparation, Lysis Solution supplemented with DNase I was used.The probes for real-time PCR used were TaqMan(R) Gene Expression Assays(CTNNB1, FAM probe) (Hs00355045_m1, manufactured by Applied Biosystems,Inc.) for the human β-catenin gene and a human GAPDH gene probe as aninternal standard (VIC probe, Hs99999905_m1, manufactured by AppliedBiosystems, Inc.). 5 μL of TaqMan(R) Fast Advanced Master Mix, 2 μL ofRNase-Free Water, 0.5 μL of each gene probe, and 2 μL of the preparedcDNA solution were added per well of a 384-well PCR plate (manufacturedby Applied Biosystems, Inc.) to bring the total amount to 10 μL, whichwas then loaded in ViiA™ 7 Real-time PCR system (manufactured by AppliedBiosystems, Inc.) and subjected to PCR under conditions given below. Thereal-time PCR was carried out at N=4 for the cDNA prepared from thelysate.

PCR initial activation: 95° C. for 20 seconds

PCR: 95° C. for 1 second

-   -   62° C. for 20 seconds

This PCR cycle was repetitively performed 40 times.

(3) Real-time PCR Analysis

The quantitative analysis was conducted by the ΔΔCt method. A value(ΔΔCt) was determined by subtracting ΔCt of an untreated cell (═NC) fromthe difference in Ct value (ΔCt) between human β-catenin and human GAPDHof each transfectant, and a relative value (RQ) to NC was calculatedaccording to the following expression:RQ=2^(−ΔΔCt)When RQ=1 was defined as 0% rate of inhibition and RQ=0 was defined astheoretical 100% rate of inhibition, the IC₅₀ value of the nucleic acidlipid particle was calculated using GraphPad PRISM (GraphPad SoftwareInc.). As a result, as shown in Table 7, the nucleic acid lipid particlecontaining the compound of Example 8 exhibited strong inhibitoryactivity against β-catenin gene expression, as compared with the nucleicacid lipid particle containing the lipid compound 1 used as a control.These results demonstrated that the compound of Example 8 is a novellipid useful for preparing nucleic acid lipid particles that exhibitstrong activity.

TABLE 7 β-catenin gene expression inhibitory activity IC50 (nM) Compound1 >30 Example 8 8.3

Test Example 4

As described below, the strength of human β-catenin gene expressioninhibitory activity was compared among nucleic acid lipid particles eachprepared using a novel lipid.

(1) Transfection

The concentration of a human uterine cervix cancer Hela cell line(derived from human uterine cervix cancer) was adjusted to 50000cells/mL in a DMEM medium (manufactured by Invitrogen Corp.) containing10% fetal bovine serum (culture medium). Then, the resulting culturesolution was inoculated at 100 μL/well to a 96-well flat-bottomed plate(manufactured by Corning Inc./Falcon) and cultured at 37° C. for 1 dayunder 5.0% CO₂. The nucleic acid lipid particle-containing dispersionprepared in Example 31 was diluted with a culture medium to preparedilution series having final double-stranded polynucleotideconcentrations of 30, 3, 0.3, and 0.03 nM in the medium. Then, eachdilution was added to the cells after removal of the culturesupernatant, and the culture was further continued for 4 hours. Thisoperation was performed at N=3 for each concentration.

(2) Real-time PCR

A lysate and cDNA for real-time PCR measurement were prepared from thetransfected cells using TaqMan(R) Fast-Cells-to-Ct kit (LifeTechnologies, Inc./Ambion) according to the instruction manual. In thelysate preparation, Lysis Solution supplemented with DNase I was used.The probes for real-time PCR used were TaqMan(R) Gene Expression Assays(CTNNB1, FAM probe) (Hs00355045_m1, manufactured by Applied Biosystems,Inc.) for the human β-catenin gene and a human GAPDH gene probe as aninternal standard (VIC probe, Hs99999905_m1, manufactured by AppliedBiosystems, Inc.). 5 μL of TaqMan(R) Fast Advanced Master Mix, 2 μL ofRNase-Free Water, 0.5 μL of each gene probe, and 2 μL of the preparedcDNA solution were added per well of a 384-well PCR plate (manufacturedby Applied Biosystems, Inc.) to bring the total amount to 10 μL, whichwas then loaded in ViiA™ 7 Real-time PCR system (manufactured by AppliedBiosystems, Inc.) and subjected to PCR under conditions given below. Thereal-time PCR was carried out at N=4 for the cDNA prepared from thelysate.

PCR initial activation: 95° C. for 20 seconds

PCR: 95° C. for 1 second

-   -   62° C. for 20 seconds

This PCR cycle was repetitively performed 40 times.

(3) Real-time PCR Analysis

The quantitative analysis was conducted by the ΔΔCt method. A value(ΔΔCt) was determined by subtracting ΔCt of an untreated cell (═NC) fromthe difference in Ct value (ΔCt) between human β-catenin and human GAPDHof each transfectant, and a relative value (RQ) to NC was calculatedaccording to the following expression:RQ=2^(−ΔΔCt)When RQ=1 was defined as 0% rate of inhibition and RQ=0 was defined astheoretical 100% rate of inhibition, the IC₅₀ value of the nucleic acidlipid particle was calculated using GraphPad PRISM (GraphPad SoftwareInc.). As a result, as shown in Table 8, the nucleic acid lipid particlecontaining the compound of Example 8 exhibited strong inhibitoryactivity against β-catenin gene expression, as compared with the nucleicacid lipid particle containing the lipid compound 1 used as a control.These results demonstrated that the compound of Example 8 is a novellipid useful for preparing nucleic acid lipid particles that exhibitstrong activity.

TABLE 8 β-catenin gene expression inhibitory activity IC50 (nM) Compound1 20 Example 8 2.2

Test Example 5 Measurement of Cell Growth Inhibitory Activity ofCompound of Example Against Hep3B Cell (Human Liver Cancer Cell)

The medium used is MEM (manufactured by Invitrogen Corp.) (containing10% fetal bovine serum (manufactured by HyClone Laboratories, Inc.), 1mM sodium pyruvate (manufactured by Invitrogen Corp.), and 1×non-essential amino acids (manufactured by Invitrogen Corp.)). Humanliver cancer cell line Hep3B cells having a given density are placed(150 μL/well) in a 96-well plate and subsequently cultured at 37° C. for24 hours under 5% CO₂. The nucleic acid lipid particle-containingdispersion prepared in Example 31 is further added at finalconcentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 μM to each well, andthe cells are subsequently cultured for 72 hours (3 days). After theculture for 72 hours (3 days), the cell growth inhibitory activity ofthe compound of each Example is measured using MTT assay. Specifically,20 μL of a MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) isfurther added to each well, and the cells are cultured at 37° C. for 4hours under 5% CO₂. After removal of the culture supernatant, DMSO (150μL) is further added to each well, followed by shaking for 5 minutes.The absorbance (540 nm) from the plate is measured using a plate reader(SpectraMax Plus³⁸⁴, manufactured by Molecular Devices Corporation). Therelative ratio between the number of live cells in the compoundadministration group and the number of live cells in an untreated cellgroup is determined. Then, the IC₅₀ concentration at which of the growthof cells is inhibited by 50% is calculated.

Test Example 6 In Vivo Antitumor Test of Compound of Example

After acclimatization and raising of each nude mouse for 1 week, 1×10⁷cultured human Hep3B cells are subcutaneously transplanted to thelateral region of the nude mouse. Approximately 2 weeks after the tumortransplantation, the mice are grouped with the tumor volume as an index,and the nucleic acid lipid particle-containing dispersion prepared inExample 31 is intravenously administered (administered at a dose such as1 or 3 mg/kg) twice or three times a week to the tail of each mouse. PBSis administered to a control group. The tumor size is measured, andchanges in tumor volume are observed.

In the case of verifying an in vivo knockdown effect, on the day afterthe administration, a tumor mass is collected from the cancer-bearingmouse, and a nucleic acid is extracted using QIAzol Lysis Reagent(manufactured by Qiagen N.V.) and chloroform. Then, total RNA ispurified using RNeasy mini kit (manufactured by Qiagen N.V.) accordingto the attached protocol. This is used to quantify the mRNA of thetarget molecule by Taqman PCR.

Example 33 Preparation of Double-stranded Polynucleotide-encapsulatedNucleic Acid Lipid Particle

A lipid solution having a total lipid concentration of 26.8 mM inethanol with distearoylphosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter, referred to asDSPC, NOF CORPORATION), cholesterol (hereinafter, referred to as Chol,Sigma-Aldrich, Inc.), the compound described in Example 8 (hereinafter,referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DMA) were prepared at a molar ratio described inTable 9.

The concentration of a double-stranded polynucleotide described inNature Biotechnology (2008) 26, 561-569 (siFVII: siRNA against mouseFactor VII) was adjusted to 1 mg/mL with a citrate buffer solution (10mM citrate buffer, pH 4.0) containing 30% ethanol to obtain adouble-stranded polynucleotide solution.

The lipid solution, the double-stranded polynucleotide solution, and acitrate buffer solution (20 mM citrate buffer, pH 4.0) were heated to37° C. The lipid solution was added dropwise to the citrate buffersolution (20 mM citrate buffer, pH 4.0) and mixed therewith such thatthe volume ratio between the lipid solution and the citrate buffersolution was 3:7 to obtain a crude liposome dispersion. Subsequently,the crude liposome dispersion was added dropwise to the double-strandedpolynucleotide solution and mixed therewith such that the ratio (N/P) ofLP-derived nitrogen atoms (N) to double-stranded polynucleotide-derivedphosphorus atoms (P) was 3. The mixture was incubated at 37° C. for 30minutes to obtain a nucleic acid lipid particle dispersion. The nucleicacid lipid particle dispersion was dialyzed against approximately 100 mLof a phosphate buffer solution (pH 7.4) for 12 to 18 hours(Float-A-Lyzer G2, MWCO: 100 kD, Spectra/Por) for the removal of ethanoland the removal of unencapsulated double-stranded polynucleotides byneutralization to obtain a purified dispersion of a nucleic acid lipidparticle containing the double-stranded polynucleotide and the lipiddescribed in Table 9.

TABLE 9 DSPC Chol LP PEG-C-DMA Particle 1 10 68 20 2 Particle 2 10 58 302 Particle 3 10 48 40 2 Particle 4 10 43 45 2 Particle 5 10 38 50 2Particle 6 10 33 55 2 Particle 7 10 28 60 2 Particle 8 10 18 70 2Particle 9 55 33 10 2 Particle 10 45 33 20 2 Particle 11 35 33 30 2Particle 12 25 33 40 2 Particle 13 20 33 45 2 Particle 14 15 33 50 2Particle 15 10 33 55 2 Particle 16 5 33 60 2 Particle 17 0 33 65 2Particle 18 10 49.5 40 0.5 Particle 19 10 49 40 1 Particle 20 10 48.5 401.5 Particle 21 10 48 40 2 Particle 22 10 47.5 40 2.5 Particle 23 10 4740 3 Particle 24 10 46.5 40 3.5 Particle 25 10 46 40 4 Particle 26 10 4540 5 Particle 27 10 40 40 10

Example 34 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

The nucleic acid lipid particle dispersion prepared in Example 33 wascharacterized. The characterization was conducted by the methodsdescribed in Example 32, and the rate of polynucleotide encapsulation inthe nucleic acid lipid particle described in Example 33, the weightratio of the polynucleotide to the lipid, and the average particle sizeare shown in Tables 10, 11, and 12.

TABLE 10 Rate of Lipid encapsulation siRNA/lipid Particle sizecomposition * (%) (wt/wt) ** (nm) Particle 1 10/68/20/2 96 0.044 133 ±23 Particle 2 10/58/30/2 98 0.061 153 ± 46 Particle 3 10/48/40/2 980.083 127 ± 20 Particle 4 10/43/45/2 98 0.084 143 ± 41 Particle 510/38/50/2 98 0.094 133 ± 27 Particle 6 10/33/55/2 94 0.115 137 ± 10Particle 7 10/28/60/2 81 0.117 184 ± 39 Particle 8 10/18/70/2 50 0.127162 ± 31 * Lipid composition: DSPC/Chol/LP/PEG-C-DMA (molar ratio) **siRNA/lipid (wt/wt): weight ratio of polynucleotide to lipid

TABLE 11 Rate of Lipid encapsulation siRNA/lipid Particle sizecomposition* (%) (wt/wt)** (nm) Particle 9 55/33/10/2 93 0.014 193 ± 42Particle 10 45/33/20/2 93 0.022 206 ± 77 Particle 11 35/33/30/2 90 0.055203 ± 73 Particle 12 25/33/40/2 93 0.084 177 ± 59 Particle 13 20/33/45/292 0.093 109 ± 22 Particle 14 15/33/50/2 92 0.073 111 ± 20 Particle 1510/33/55/2 95 0.111 119 ± 13 Particle 16 5/33/60/2 95 0.136 135 ± 15Particle 17 0/33/65/2 91 0.160 124 ± 25 *Lipid composition:DSPC/Chol/LP/PEG-C-DMA (molar ratio) **siRNA/lipid (wt/wt): weight ratioof polynucleotide to lipid

TABLE 12 Rate of Lipid encapsulation siRNA/lipid Particle sizecomposition* (%) (wt/wt)** (nm) Particle 18 10/49.5/40/0.5 97 0.096  349± 222 Particle 19 10/49/40/1 96 0.091 206 ± 54 Particle 2010/48.5/40/1.5 97 0.098 140 ± 54 Particle 21 10/48/40/2 98 0.091 109 ±36 Particle 22 10/47.5/40/2.5 99 0.090 140 ± 19 Particle 23 10/47/40/398 0.089 152 ± 27 Particle 24 10/46.5/40/3.5 98 0.084 131 ± 52 Particle25 10/46/40/4 98 0.086 156 ± 66 Particle 26 10/45/40/5 94 0.083  79 ± 27Particle 27 10/40/40/10 91 0.055  83 ± 47 *Lipid composition:DSPC/Chol/LP/PEG-C-DMA (molar ratio) **siRNA/lipid (wt/wt): weight ratioof polynucleotide to lipid

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 80 nm to approximately 300nm.

Test Example 7 Factor VII (FVII) Protein Measurement

The Factor VII protein was measured according to a method described inNature Biotechnology (2010) 28, 172-176. C57BL6/J mice (male, 9 weeksold) were randomly grouped (n=4). The nucleic acid lipid particledispersion prepared in Example 33 was intravenously injected at a doseof 0.3 mg/kg to the tail of each mouse. One day after theadministration, approximately 50 μL of blood was collected from the tailvein, and plasma was obtained. The amount of the Factor VII protein inthe obtained plasma was measured using Biophen FVII assay kit(manufactured by Aniara Corp.) according to the attached protocol.

When the amount of FVII of respective plasma samples collected in equalamounts from individuals in a PBS administration group was defined as100%, the relative ratio (%) of the amount of FVII in a plasma sample ofeach individual was used as a measurement value (A). An average value(B) was determined from the respective measurement values of theindividuals in the PBS administration group. The relative ratio of themeasurement value (A) of each individual was determined from theexpression: A/B×100 (%). The average value of the relative ratios in theadministration group of each nucleic acid lipid particle is shown inTables 13, 14, and 15. As a result, as shown in Tables 13, 14, and 15,particles 1 to 8, particles 12 to 17, particles 20 to 24, particle 26,and particle 27 as the nucleic acid lipid particles prepared in Example33 exhibited strong FVII inhibitory activity. These results demonstratedthat a nucleic acid lipid particle having lipid composition as found inparticles 1 to 8, particles 12 to 17, particles 20 to 24, particle 26,and particle 27 is useful as a nucleic acid lipid particle capable ofinhibiting gene expression.

TABLE 13 Relative amount of FVII (%) PBS 100 Particle 1 36 Particle 2<10 Particle 3 <10 Particle 4 20 Particle 5 <10 Particle 6 <10 Particle7 <10 Particle 8 <10

TABLE 14 Relative amount of FVII (%) PBS 100 Particle 10 No activityParticle 11 No activity Particle 12 79 Particle 13 22 Particle 14 <10Particle 15 26 Particle 16 <10 Particle 17 <10

TABLE 15 Relative amount of FVII (%) PBS 100 Particle 18 No activityParticle 19 No activity Particle 20 <10 Particle 21 <10 Particle 22 <10Particle 23 <10 Particle 24 27 Particle 26 28 Particle 27 49

Reference Example 46 3-Bromopropyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate

A solution of (19Z,22Z)-octacosa-19,22-dien-11-ol (0.55 g, 1.4 mmol)obtained in Reference Example 17 and pyridine (0.67 g, 8.5 mmol) intoluene (14 mL) was cooled to 0° C. in an ice bath, and a solution oftriphosgene (0.28 g, 0.93 mmol) in toluene (2.0 mL) was added theretoover 2 minutes. After stirring at 0° C. for 15 minutes, the reactionmixture was heated to room temperature, stirred at for 1 hour, andcooled to 0° C. again. 3-Bromo-1-propanol (2.0 g, 14 mmol) was addedthereto, and the mixture was reacted overnight at room temperature.After treatment with a saturated aqueous solution of sodium bicarbonate,the reaction mixture was subjected to extraction with ethylacetate-hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.32 g, 41%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.21-1.40 (32H, m),1.52-1.61 (4H, m), 2.01-2.09 (4H, m), 2.23 (2H, quint, J=6.3 Hz), 2.77(2H, t, J=6.6 Hz), 3.49 (2H, t, J=6.3 Hz), 4.27 (2H, t, J=6.3 Hz),4.66-4.73 (1H, m), 5.29-5.44 (4H, m).

Example 35 3-(Azetidin-1-yl)propyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (Exemplary Compound 1-76)

To a solution of 3-bromopropyl(9Z,12Z)-octacosa-19,22-dien-11-ylcarbonate (0.16 g, 0.28 mmol) obtained in Reference Example 46 intetrahydrofuran (8.0 mL), azetidine (0.40 g, 7.0 mmol) was added, andthe mixture was reacted at 120° C. for 50 minutes under microwaveirradiation. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction with ethylacetate-hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (119 mg, 77%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.21-1.40 (32H, m),1.47-1.61 (4H, m), 1.71 (2H, tt, J=6.6, 7.4 Hz), 2.01-2.10 (m, 6H), 2.46(2H, t, J=7.4 Hz), 2.77 (2H, t, J=6.6 Hz), 3.16 (4H, t, J=6.6 Hz), 4.15(2H, t, J=6.6 Hz), 4.64-4.71 (1H, m), 5.28-5.42 (4H, m).

MS (ESI+) m/z 548 [M+H]⁺

HRMS (ESI+) m/z 548.5044 (0.1 mDa).

Example 36(1-Methylpiperidin-3-yl)methyl(6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ylcarbonate (Exemplary Compound 2-102)

A solution of (6Z,9Z,26Z,29Z)-pentatriaconta-6,9,26,29-tetraen-18-ol(0.22 g, 0.44 mmol) obtained in Reference Example 4 and pyridine (0.22g, 2.8 mmol) in toluene (4.4 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.090 g, 0.30 mmol) in toluene (0.66 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 15 minutes,the reaction mixture was heated to room temperature, stirred for 1 hour,and cooled to 0° C. again. (1-Methyl-3-piperidyl)methanol (0.60 g, 4.6mmol) was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction with ethylacetate-hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (201 mg, 70%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, m), 0.92-1.04 (1H, m), 1.21-1.40(36H, m), 1.48-1.78 (8H, m), 1.85-1.94 (1H, m), 1.96-2.10 (9H, m), 2.26(3H, s), 2.77 (4H, t, J=6.6 Hz), 2.72-2.90 (2H, m), 3.94 (1H, dd, J=7.4,10.6 Hz), 4.05 (1H, dd, J=5.5, 10.6 Hz), 4.64-4.72 (1H, m), 5.28-5.42(8H, m).

MS (ESI+) m/z 656 [M+H]⁺

HRMS (ESI+) m/z 656.5981 (−0.1 mDa).

Example 373-(Dimethylamino)propyl(19Z,22R)-22-(tetrahydro-2H-pyran-2-yloxy)octacos-19-en-11-ylcarbonate (Exemplary Compound 1-334)

To a solution of3-(dimethylamino)propyl(19Z,22R)-22-hydroxyoctacos-19-en-11-yl carbonate(0.21 g, 0.38 mmol) obtained in Example 27 and p-toluenesulfonic acidmonohydrate (0.079 g, 0.42 mmol) in dichloromethane (3.8 mL),3,4-dihydro-2H-pyran (0.16 g, 1.9 mmol) was added, and the mixture wasreacted at room temperature for 3 hours. After treatment with asaturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction, and the obtained organic layer was driedover anhydrous magnesium sulfate. The solvent was distilled off underreduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (210 mg, 87%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.85 (48H, m), 1.84(2H, tt, J=6.7, 7.4 Hz), 1.98-2.06 (2H, m), 2.22 (6H, s), 2.36 (2H, t,J=7.4 Hz), 3.44-3.52 (1H, m), 3.57-3.72 (1H, m), 3.87-3.97 (1H, m), 4.18(2H, t, J=6.7 Hz), 4.64-4.75 (2H, m), 5.32-5.50 (2H, m).

MS (ESI+) m/z 638 [M+H]⁺

HRMS (ESI+) m/z 638.5723 (1.6 mDa).

Reference Example 47 (19Z,22R)-22-Hydroxyoctacos-19-en-11-one

To (19Z,22R)-22-{[tert-butyl(dimethyl)silyl]oxy}octacos-19-en-11-one(2.0 g, 3.7 mmol) obtained in Reference Example 38, a 1 M solution oftetra-n-butylammonium fluoride in tetrahydrofuran was added, and themixture was reacted at room temperature for 5 hours. After watertreatment to terminate the reaction, the reaction mixture was subjectedto extraction with hexane, and the obtained organic layer was subjectedto silica gel column chromatography to obtain a liquid containing thecompound of interest (0.66 g). The product was used directly in the nextreaction without being further purified.

Reference Example 48 (7R,9Z)-18-Hydroxyoctacos-9-en-7-yl acetate

To a solution of the mixture containing(19Z,22R)-22-hydroxyoctacos-19-en-11-one (0.33 g) obtained in ReferenceExample 47 and pyridine (1.1 g, 14 mmol) in dichloromethane (7.1 mL),acetyl chloride (0.56 g, 7.1 mmol) was added dropwise over 1 minute, andthe mixture was reacted at room temperature for 1 hour. After watertreatment to terminate the reaction, extraction was carried out, andvolatile matter was removed under reduced pressure to obtain a liquidmixture. This mixture was dissolved in tetrahydrofuran (2.1 mL) andmethanol (2.1 mL). To the solution, sodium borohydride (0.054 g, 1.4mmol) was added, and the mixture was reacted at room temperature for 1hour. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was subjected to silica gelcolumn chromatography to obtain a liquid containing the compound ofinterest (0.33 g). The product was used directly in the next reactionwithout being further purified.

Example 38(7R,9Z)-18-([{(1-Methylpiperidin-3-yl)methoxy]carbonyl}oxy}octacos-9-en-7-ylacetate (Exemplary Compound 2-132)

A solution of the mixture containing (7R,9Z)-18-hydroxyoctacos-9-en-7-ylacetate (0.33 g) obtained in Reference Example 48 and pyridine (0.35 g,4.5 mmol) in toluene (7.1 mL) was cooled to 0° C. in an ice bath, and asolution of triphosgene (0.14 g, 0.49 mmol) in toluene (1.1 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 10 minutes, thereaction mixture was heated to room temperature, stirred for 30 minutes,and cooled to 0° C. again. (1-Methyl-3-piperidyl)methanol (0.96 g, 7.4mmol) was added thereto, and the mixture was reacted at room temperaturefor 90 minutes. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith ethyl acetate, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(110 mg).

¹H-NMR (400 MHz, CDCl₃) δ: 0.86-0.91 (6H, m), 0.93-1.05 (1H, m),1.17-1.38 (36H, m), 1.48-1.78 (8H, m), 1.85-1.94 (1H, m), 1.96-2.07 (6H,m), 2.24-2.34 (5H, m), 2.74 (1H, d, J=10.5 Hz), 2.86 (1H, d, J=10.5 Hz),3.95 (1H, ddd, J=2.7, 7.4, 10.2 Hz), 4.06 (1H, ddd, J=2.7, 5.9, 10.2Hz), 4.64-4.71 (1H, m), 4.87 (1H, quint, J=6.3 Hz), 5.27-5.37 (1H, m),5.43-5.51 (1H, m).

MS (ESI+) m/z 622 [M+H]⁺

HRMS (ESI+) m/z 622.5438 (2.8 mDa).

Reference Example 49 (7R,9Z)-18-Hydroxyoctacos-9-en-7-yl caproate

To a solution of the mixture containing(19Z,22R)-22-hydroxyoctacos-19-en-11-one (0.30 g) obtained in ReferenceExample 47 and pyridine (1.1 g, 14 mmol) in dichloromethane (7.1 mL),caproic anhydride (0.76 g, 3.5 mmol) was added dropwise over 1 minute,then 4-(dimethylamino)pyridine (0.01 g) was added, and the mixture wasreacted at room temperature for 90 minutes. After treatment with asaturated aqueous solution of sodium bicarbonate, extraction was carriedout, and volatile matter was removed under reduced pressure to obtain aliquid mixture. This mixture was dissolved in tetrahydrofuran (2.1 mL)and methanol (2.1 mL). To the solution, sodium borohydride (0.054 g, 1.4mmol) was added, and the mixture was reacted at room temperature for 4hours. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was subjected to silica gelcolumn chromatography to obtain a liquid containing the compound ofinterest (0.17 g). The product was used directly in the next reactionwithout being further purified.

Example 39(7R,9Z)-18-({[3-(Dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-ylcaproate (Exemplary Compound 1-255)

A solution of the mixture containing (7R,9Z)-18-hydroxyoctacos-9-en-7-ylcaproate (0.17 g) obtained in Reference Example 49 and pyridine (0.16 g,2.0 mmol) in toluene (3.3 mL) was cooled to 0° C. in an ice bath, and asolution of triphosgene (0.067 g, 0.22 mmol) in toluene (0.49 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 15 minutes, thereaction mixture was heated to room temperature, stirred for 45 minutes,and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.35 g, 3.4 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (150 mg).

¹H-NMR (400 MHz, CDCl₃) δ: 0.84-0.92 (9H, m), 1.20-1.38 (40H, m),1.48-1.66 (8H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.97-2.04 (2H, m), 2.22(6H, s), 2.27 (2H, t, J=7.4 Hz), 2.36 (2H, t, J=7.4 Hz), 4.18 (2H, t,J=6.6 Hz), 4.64-4.72 (1H, m), 4.88 (1H, quint, J=6.3 Hz), 5.27-5.37 (1H,m), 5.42-5.51 (1H, m).

MS (ESI+) m/z 652 [M+H]⁺

HRMS (ESI+) m/z 652.5892 (1.2 mDa).

Example 403-(Dimethylamino)propyl(19Z,22R)-22-(tetrahydrofuran-2-yloxy)octacos-19-en-11-ylcarbonate (Exemplary Compound 1-377)

To a solution of3-(dimethylamino)propyl(19Z,22R)-22-hydroxyoctacos-19-en-11-yl carbonate(0.055 g, 0.099 mmol) obtained in Example 27 and p-toluenesulfonic acidhydrate (0.026 g, 0.14 mmol) in dichloromethane (1.3 mL),2,3-dihydrofuran (0.044 g, 0.63 mmol) was added, and the mixture wasreacted overnight at room temperature. After treatment with a saturatedaqueous solution of sodium bicarbonate, the reaction mixture wassubjected to extraction with dichloromethane, and the obtained organiclayer was dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the residue was then subjectedto silica gel column chromatography to obtain the compound of interestas a colorless liquid (33 mg, 53%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.82-0.88 (6H, m), 1.20-1.62 (48H, m),1.74-2.05 (8H, m), 2.21 (6H, s), 2.33 (2H, t, J=7.4 Hz), 3.51-4.05 (3H,m), 4.15 (2H, t, J=6.7 Hz), 4.62-4.70 (1H, m), 5.28-5.46 (3H, m).

MS (ESI+) m/z 624 [M+H]⁺

HRMS (ESI+) m/z 624.5574 (0.7 mDa).

Example 41(7R,9Z,26Z,29R)-18-({[3-(Dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyldipropionate

To a solution of3-(dimethylamino)propyl(7R,9Z,26Z,29R)-7,29-dihydroxypentatriaconta-9,26-dien-18-ylcarbonate (0.35 g, 0.53 mmol) obtained in Example 22 and pyridine (0.83g, 10.5 mmol) in dichloromethane (5.3 mL), propionic acid chloride (0.49g, 5.3 mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withdichloromethane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (260 mg, 64%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.84-0.91 (6H, m), 1.13 (6H, t, J=7.4 Hz),1.22-1.39 (36H, m), 1.48-1.61 (8H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz),2.01 (4H, dd, J=6.6, 7.0 Hz), 2.22 (6H, s), 2.30 (4H, q, J=7.4 Hz),2.28-2.33 (4H, m), 2.36 (2H, t, J=7.4 Hz), 4.18 (2H, t, J=6.6 Hz),4.64-4.72 (1H, m), 4.88 (2H, tt, J=5.9, 6.6 Hz), 5.33 (2H, ttd, J=1.2,7.0, 10.9 Hz), 5.46 (2H, ttd, J=1.2, 7.0, 10.9 Hz).

MS (ESI+) m/z 778 [M+H]⁺

HRMS (ESI+) m/z 778.6555 (−0.6 mDa).

Reference Example 50 (9Z,12R)-Octadec-9-ene-1,12-diol

To a solution of lithium aluminum hydride (3.87 g, 102 mmol) intetrahydrofuran (235 mL), methyl (9Z,12R)-12-hydroxyoctadec-9-enoate(31.1 g, 78.4 mmol, compound known by the literature (J. Org. Chem.,2001, 66, 22, 7487-7495)) was added dropwise over 50 minutes, and themixture was reacted at room temperature for 1.5 hours. After treatmentwith water (4 mL), ethyl acetate was added thereto, and solid componentswere filtered off. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (17.9 g, 62%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=6.6 Hz), 1.21-1.87 (22H, m),1.98-2.07 (2H, m), 2.20-2.40 (2H, m), 3.44-3.52 (1H, m), 3.58-3.71 (3H,m), 3.87-3.97 (1H, m), 4.64-4.74 (1H, m), 5.33-5.49 (2H, m).

Reference Example 51 (9Z,12R)-12-Hydroxyoctadec-9-en-1-ylmethanesulfonate

A solution of (9Z,12R)-octadec-9-ene-1,12-diol (17.9 g, 48.6 mmol)obtained in Reference Example 50 and triethylamine (5.90 g, 58.3 mmol)in dichloromethane (122 mL) was cooled to 0° C. Methanesulfonyl chloride(6.68 g, 58.3 mmol) was added dropwise thereto over 5 minutes, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with dichloromethane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure to obtain a yellow liquidcontaining the compound of interest.

Reference Example 52 (7R,9Z)-18-Bromooctadec-9-en-7-ol

To a solution of the liquid containing(9Z,12R)-12-hydroxyoctadec-9-en-1-yl methanesulfonate (21.6 g, 48.4mmol, theoretical amount) obtained in Reference Example 51 in diethylether (145 mL), a magnesium bromide-diethyl ether complex (31.2 g, 121mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with ice water, the reaction mixture wassubjected to extraction with diethyl ether, and the obtained organiclayer was dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the residue was then dissolvedin ethanol (100 mL). To the solution, a 2 N aqueous hydrochloric acidsolution (80 mL) was added, and the mixture was stirred overnight atroom temperature. Volatile matter was distilled off under reducedpressure. The residue was subjected to extraction with ethyl acetate,and the obtained organic layer was dried over anhydrous magnesiumsulfate. The solvent was distilled off under reduced pressure, and theresidue was then subjected to silica gel column chromatography to obtainthe compound of interest as a colorless liquid (14.1 g, 84%).

Reference Example 532-{[(7R,9Z)-18-Bromooctadec-9-en-7-yl]oxy}tetrahydro-2H-pyran

To a solution of (7R,9Z)-18-bromooctadec-9-en-7-ol (14.1 g, 40.6 mmol)obtained in Reference Example 52 and p-toluenesulfonic acid hydrate(0.154 g, 0.81 mmol) in dichloromethane (81.2 mL), 3,4-dihydro-2H-pyran(6.83 g, 81.2 mmol) was added, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith dichloromethane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(15.1, 86%).

Reference Example 54(7R,9Z,28Z,31R)-7,31-Bis(tetrahydro-2H-pyran-2-yloxy)heptatriaconta-9,28-dien-19-ol

Dried magnesium (shavings, 0.42 g, 17.4 mmol) was dipped intetrahydrofuran (3.0 mL). 1,2-Dibromoethane (3 drops) was added thereto,and the mixture was vigorously stirred. After the solution turnedblack-gray, a solution of2-{[(7R,9Z)-18-bromooctadec-9-en-7-yl]oxy}tetrahydro-2H-pyran (5.00 g,11.6 mmol) obtained in Reference Example 53 in tetrahydrofuran (8.5 mL)was added thereto over 1 hour, and the mixture was stirred at roomtemperature for 3 hours. To this solution, ethyl formate (0.47 g, 5.79mmol) was added, and the mixture was reacted overnight at roomtemperature. After water treatment to terminate the reaction, thereaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen dissolved in ethanol (25 mL). To the solution, a 5 N aqueous sodiumhydroxide solution (5 mL) was added, and the mixture was stirred at roomtemperature for 1 hour. The reaction mixture was diluted with water andthen subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (2.10 g, 50%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.23-1.62 (52H, m),1.66-1.89 (4H, m), 1.98-2.07 (4H, m), 2.20-2.41 (4H, m), 3.45-3.52 (2H,m), 3.54-3.71 (3H, m), 3.87-3.98 (2H, m), 4.64-4.75 (2H, m), 5.33-5.50(4H, m).

Reference Example 55(7R,9Z,28Z,31R)-7,31-Bis(tetrahydro-2H-pyran-2-yloxy)heptatriaconta-9,28-dien-19-yl3-(dimethylamino)propyl

A solution of(7R,9Z,28Z,31R)-7,31-bis(tetrahydro-2H-pyran-2-yloxy)heptatriaconta-9,28-dien-19-ol(1.00 g, 1.36 mmol) obtained in Reference Example 54 and pyridine (0.68g, 8.6 mmol) in toluene (13.6 mL) was cooled to 0° C. in an ice bath,and a solution of triphosgene (0.279 g, 0.94 mmol) in toluene (2.05 mL)was added thereto over 2 minutes. After stirring at 0° C. for 15minutes, the reaction mixture was heated to room temperature, stirredfor 1.5 hours, and cooled to 0° C. again. 3-Dimethylamino-1-propanol(1.48 g, 14.3 mmol) was added thereto, and the mixture was reactedovernight at room temperature. After treatment with a saturated aqueoussolution of sodium bicarbonate, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(1.00 g, 85%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.85 (56H, m),1.79-1.88 (2H, m), 1.98-2.06 (4H, m), 2.20-2.38 (12H, m), 3.44-3.52 (2H,m), 3.58-3.71 (2H, m), 3.87-3.97 (2H, m), 4.18 (2H, t, J=6.7 Hz),4.64-4.74 (3H, m), 5.32-5.49 (4H, m).

Reference Example 56(7R,9Z,28Z,31R)-7,31-Dihydroxyheptatriaconta-9,28-dien-19-yl3-(dimethylamino)propylcarbonate

To a solution of(7R,9Z,28Z,31R)-7,31-bis(tetrahydro-2H-pyran-2-yloxy)heptatriaconta-9,28-dien-19-yl3-(dimethylamino)propyl(1.00 g, 1.16 mmol) obtained in Reference Example 55 in ethanol (11.6mL), a 2 N aqueous hydrochloric acid solution (5.79 mL, 11.6 mmol) wasadded, and the mixture was reacted at room temperature for 2.5 hours.After treatment with a saturated aqueous solution of sodium bicarbonate,the reaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (0.68 g, 84%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.61 (48H, m), 1.84(2H, tt, J=6.6, 7.4 Hz), 2.01-2.08 (4H, m), 2.18-2.24 (10H, m), 2.34(2H, t, J=7.4 Hz), 3.61 (2H, quint, J=5.9 Hz), 4.18 (2H, t, J=6.6 Hz),4.64-4.72 (1H, m), 5.36-5.44 (2H, m), 5.52-5.61 (2H, m).

Example 42(7R,9Z,28Z,31R)-19-({[3-(Dimethylamino)propoxy]carbonyl}oxy)heptatriaconta-9,28-diene-7,31-diyldiacetate

To a solution of(7R,9Z,28Z,31R)-7,31-dihydroxyheptatriaconta-9,28-dien-19-yl3-(dimethylamino)propyl carbonate (0.68 g, 0.98 mmol) obtained inReference Example 56 and pyridine (2.3 g, 29 mmol) in dichloromethane(9.8 mL), acetyl chloride (1.2 g, 15 mmol) was added, and the mixturewas reacted overnight at room temperature. After treatment with asaturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with dichloromethane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain the compound ofinterest as a colorless liquid (523 mg, 69%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.20-1.38 (40H, m),1.48-1.61 (8H, m), 1.85 (2H, tt, J=6.6, 7.4 Hz), 1.98-2.08 (10H, m),2.22 (6H, s), 2.29 (4H, dt, J=6.3, 6.6 Hz), 2.36 (2H, t, J=7.4 Hz), 4.18(2H, t, J=6.6 Hz), 4.64-4.72 (1H, m), 4.87 (2H, quint, J=6.3 Hz), 5.36(1H, ttd, J=1.2, 6.6, 10.9 Hz), 5.47 (1H, ttd, J=1.2, 6.6, 10.9 Hz).

MS (ESI+) m/z 778 [M+H]⁺

HRMS (ESI+) m/z 778.6557 (−0.4 mDa).

Reference Example 57(21Z,24R)-24-{[tert-Butyl(dimethyl)silyl]oxy}triacont-21-en-13-one

To a solution of(9Z,12R)-12-{[tert-butyl(dimethyl)silyl]oxy}-N-methoxy-N-methyloctadec-9-enamide(0.80 g, 1.8 mmol) obtained in Reference Example 32 in diethyl ether(8.8 mL), a solution of 1 N n-dodecyl magnesium bromide in diethyl ether(2.6 mL, 2.6 mmol) was added dropwise over 1 minute, and the mixture wasthen reacted overnight at room temperature. After treatment with asaturated aqueous solution of ammonium chloride, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (0.55 g, 55%).

Reference Example 58 (21Z,24R)-24-Hydroxytriacont-21-en-13-one

To (21Z,24R)-24-{[tert-butyl(dimethyl)silyl]oxy}triacont-21-en-13-one(0.55 g, 0.97 mmol) obtained in Reference Example 57, a solution of 1 Ntetra-n-butylammonium fluoride in tetrahydrofuran (4.9 mL, 4.9 mmol) wasadded, and the mixture was reacted overnight at room temperature. Aftertreatment with water, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.23 g, 52%).

Reference Example 59 (7R,9Z)-18-Oxotriacont-9-en-7-yl acetate

To a solution of (21Z,24R)-24-hydroxytriacont-21-en-13-one (0.23 g, 0.51mmol) obtained in Reference Example 58 and pyridine (0.81 g, 10 mmol) indichloromethane (5.1 mL), acetyl chloride (0.40 g, 5.1 mmol) was added,and the mixture was reacted overnight at room temperature. Aftertreatment with a saturated aqueous solution of sodium bicarbonate, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (0.21 g, 84%).

Reference Example 60 (7R,9Z)-18-Hydroxytriacont-9-en-7-yl acetate

To a solution of (7R,9Z)-18-oxotriacont-9-en-7-yl acetate (0.21 g, 0.43mmol) obtained in Reference Example 59 in tetrahydrofuran (1.3 mL) andmethanol (1.3 mL), sodium borohydride (32 mg, 0.85 mmol) was added, andthe mixture was reacted at room temperature for 1 hour. After treatmentwith a saturated aqueous solution of ammonium chloride, the reactionmixture was subjected to extraction with ethyl acetate, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure to obtain a yellow liquidcontaining the compound of interest.

Example 43(7R,9Z)-18-({[3-(Dimethylamino)propoxy]carbonyl}oxy)triacont-9-en-7-ylacetate

A solution of the liquid containing (7R,9Z)-18-hydroxytriacont-9-en-7-ylacetate (0.21 g, 0.42 mmol, theoretical amount) obtained in ReferenceExample 60 and pyridine (0.21 g, 2.7 mmol) in toluene (4.2 mL) wascooled to 0° C. in an ice bath, and a solution of triphosgene (0.087 g,0.29 mmol) in toluene (0.64 mL) was added thereto over 2 minutes. Afterstirring at 0° C. for 15 minutes, the reaction mixture was heated toroom temperature, stirred for 1.5 hours, and cooled to 0° C. again.3-Dimethylamino-1-propanol (0.46 g, 4.5 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (111 mg, 42%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.23-1.35 (36H, m),1.48-1.66 (6H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.97-2.05 (2H, m), 2.03(3H, s), 2.22 (3H, s), 2.25-2.32 (2H, m), 2.36 (2H, t, J=7.4 Hz), 4.18(2H, t, J=6.6 Hz), 4.64-4.72 (1H, m), 4.86 (1H, quint, J=6.3 Hz),5.28-5.51 (2H, m).

MS (ESI+) m/z 624 [M+H]⁺

HRMS (ESI+) m/z 624.5566 (−0.1 mDa).

Reference Example 61 (7R,9Z)-18-Oxotriacont-9-en-7-yl butyrate

To a solution of (19Z,22R)-22-hydroxyoctacos-19-en-11-one (0.34 g, 0.80mmol) obtained in Reference Example 39 and pyridine (1.3 g, 16 mmol) indichloromethane (8.0 mL), butyric acid chloride (0.86 g, 8.0 mmol) wasadded, and the mixture was reacted overnight at room temperature. Aftertreatment with a saturated aqueous solution of sodium bicarbonate, thereaction mixture was subjected to extraction with dichloromethane, andthe obtained organic layer was dried over anhydrous magnesium sulfate.The solvent was distilled off under reduced pressure, and the residuewas then subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (0.40 g, 100%).

Reference Example 62 (7R,9Z)-18-Hydroxyoctacos-9-en-7-yl butyrate

To a solution of (7R,9Z)-18-oxotriacont-9-en-7-yl butyrate (0.40 g, 0.81mmol) obtained in Reference Example 61 in tetrahydrofuran (2.4 mL) andmethanol (2.4 mL), sodium borohydride (61 mg, 1.6 mmol) was added, andthe mixture was reacted at room temperature for 1 hour. After treatmentwith a saturated aqueous solution of ammonium chloride, the reactionmixture was subjected to extraction with ethyl acetate, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure to obtain a yellow liquidcontaining the compound of interest.

Example 44(7R,9Z)-18-({[3-(Dimethylamino)propoxy]carbonyl}oxy)octacos-9-en-7-ylbutyrate

A solution of the liquid containing (7R,9Z)-18-hydroxyoctacos-9-en-7-ylbutyrate (0.40 g, 0.81 mmol, theoretical amount) obtained in ReferenceExample 62 and pyridine (0.40 g, 5.1 mmol) in toluene (8.1 mL) wascooled to 0° C. in an ice bath, and a solution of triphosgene (0.17 g,0.56 mmol) in toluene (1.2 mL) was added thereto over 2 minutes. Afterstirring at 0° C. for 15 minutes, the reaction mixture was heated toroom temperature, stirred for 1.5 hours, and cooled to 0° C. again.3-Dimethylamino-1-propanol (0.88 g, 8.5 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (99 mg, 20%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.90 (6H, m), 0.94 (3H, t, J=7.4 Hz),1.21-1.39 (32H, m), 1.47-1.62 (6H, m), 1.65 (2H, sext, J=7.4 Hz), 1.84(2H, tt, J=6.6, 7.4 Hz), 2.01 (2H, q, J=6.6 Hz), 2.22 (6H, s), 2.23-2.39(4H, m), 4.18 (2H, t, J=6.6 Hz), 4.64-4.72 (1H, m), 4.89 (2H, quint,J=6.3 Hz), 5.28-5.50 (2H, m).

MS (ESI+) m/z 624 [M+H]⁺

HRMS (ESI+) m/z 624.5599 (3.2 mDa).

Reference Example 63 Triacontan-11-ol

To a solution of icosanal (0.64 g, 2.2 mmol) in diethyl ether (11 mL), asolution of 1 N n-decyl magnesium bromide in diethyl ether (3.2 mL, 3.2mmol) was added dropwise over 2 minutes, and the mixture was thenreacted overnight at room temperature. After treatment with a 1 Naqueous hydrochloric acid solution, the reaction mixture was subjectedto extraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(0.37 g, 39%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0), 1.20-1.33 (50H, m),1.36-1.48 (4H, m), 3.54-3.62 (1H, br).

Example 45 3-(Dimethylamino)propyl triacontan-11-yl carbonate

To a solution of triacontan-11-ol (0.18 g, 0.41 mmol) obtained inReference Example 63 and pyridine (0.20 g, 2.6 mmol) in toluene (4.1mL), a solution of triphosgene (0.084 g, 0.28 mmol) in toluene (0.62 mL)was added over 2 minutes. After stirring at room temperature for 1 hour,3-dimethylamino-1-propanol (0.44 g, 4.3 mmol) was added thereto, and themixture was reacted overnight at room temperature. After treatment witha saturated aqueous solution of sodium bicarbonate, the reaction mixturewas subjected to extraction with hexane, and the obtained organic layerwas dried over anhydrous magnesium sulfate. The solvent was distilledoff under reduced pressure, and the residue was then subjected to silicagel column chromatography to obtain the compound of interest as acolorless liquid (185 mg, 79%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.84 (6H, t, J=7.0 Hz), 1.16-1.32 (50H, m),1.44-1.60 (4H, m), 1.82 (2H, tt, J=6.6, 7.4 Hz), 2.19 (6H, s), 2.32 (2H,t, J=7.4 Hz), 4.14 (2H, t, J=6.6 Hz), 4.61-4.68 (1H, m).

MS (ESI+) m/z 568 [M+H]⁺

HRMS (ESI+) m/z 568.5663 (−0.6 mDa).

Example 46 (1-Methylpiperidin-3-yl)methyl triacontan-11-yl carbonate

To a solution of triacontan-11-ol (0.18 g, 0.41 mmol) obtained inReference Example 63 and pyridine (0.20 g, 2.6 mmol) in toluene (4.1mL), a solution of triphosgene (0.084 g, 0.28 mmol) in toluene (0.62 mL)was added over 2 minutes. After stirring at room temperature for 1 hour,1-methyl-3-piperidinemethanol (0.56 g, 4.3 mmol) was added thereto, andthe mixture was reacted overnight at room temperature. After treatmentwith a saturated aqueous solution of sodium bicarbonate, the reactionmixture was subjected to extraction with hexane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain the compound ofinterest as a colorless liquid (145 mg, 60%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.20-1.36 (50H, m),1.48-1.77 (8H, m), 1.85-1.94 (1H, m), 1.95-2.07 (1H, m), 2.26 (3H, s),2.75 (1H, d, J=10.9 Hz), 2.86 (1H, d, J=10.9 Hz), 3.94 (1H, dd, J=7.4,10.9 Hz), 4.06 (1H, dd, J=5.9, 10.9 Hz), 4.64-4.71 (1H, m).

MS (ESI+) m/z 594 [M+H]⁺

HRMS (ESI+) m/z 594.5827 (0.2 mDa).

Reference Example 64 (6Z,9Z)-18-[2-(Ethenyloxy)ethoxy]octadeca-6,9-diene

To a solution of linoleyl alcohol (3.90 g, 14.6 mmol), tosylvinylethylene glycol (4.76 g, 16.1 mmol) and tetrabutylammonium sulfate (1.24g, 3.66 mmol) in toluene (23.4 mL), a 50% aqueous sodium hydroxidesolution (11.5 mL) was added, and the mixture was reacted overnight atroom temperature. Tosylvinyl ethylene glycol (2.00 g, 6.76 mmol) wasadded thereto, and the mixture was reacted at room temperature for 5days and nights. The reaction mixture was diluted with water and thensubjected to extraction with diethyl ether, and the obtained organiclayer was dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the residue was then subjectedto silica gel column chromatography to obtain the compound of interestas a yellow liquid (2.44, 50%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 1.25-1.40 (16H, m),1.54-1.63 (2H, m), 2.01-2.08 (4H, m), 2.77 (2H, t, J=6.6 Hz), 3.48 (2H,t, J=6.6 Hz), 3.64-3.68 (2H, m), 3.81-3.85 (2H, m), 4.01 (1H, dd, J=2.0,7.0 Hz), 4.19 (1H, dd, J=2.0, 14.1 Hz), 6.52 (1H, dd, J=7.0, 14.1 Hz).

Reference Example 65 2-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]ethanol

To a solution of (6Z,9Z)-18-[2-(ethenyloxy)ethoxy]octadeca-6,9-diene(5.70 g, 17 mmol) obtained in Reference Example 64 in ethanol (34 mL)and tetrahydrofuran (34 mL), a 1 N aqueous hydrochloric acid solution(17 mL, 17 mmol) was added, and the mixture was reacted at roomtemperature for 1.5 hours. The reaction mixture was diluted with waterand then subjected to extraction with hexane, and the obtained organiclayer was dried over anhydrous magnesium sulfate. The solvent wasdistilled off under reduced pressure, and the residue was then subjectedto silica gel column chromatography to obtain the compound of interestas a colorless liquid (2.00 g, 38%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (3H, t, J=7.0 Hz), 1.23-1.40 (16H, m),1.51-1.63 (2H, m), 1.95 (1H, t, J=6.3 Hz), 2.01-2.09 (4H, m), 2.77 (2H,t, J=6.6 Hz), 3.53 (2H, t, J=4.3 Hz), 3.73 (2H, dt, J=6.3, 4.3 Hz),5.29-5.43 (4H, m).

Reference Example 66 [(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]acetaldehyde

To a solution of 2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]ethanol (1.00 g,3.2 mmol) obtained in Reference Example 65 and triethylamine (1.63 g,16.1 mmol) in dimethyl sulfoxide (6.4 mL), a sulfur trioxide-pyridinecomplex (1.54 g, 9.7 mmol) was added, and the mixture was reacted atroom temperature for 1.5 hours. The reaction mixture was diluted withwater and then subjected to extraction with hexane, and the obtainedorganic layer was dried over anhydrous magnesium sulfate. The solventwas distilled off under reduced pressure, and the residue was thensubjected to silica gel column chromatography to obtain the compound ofinterest as a yellow liquid (0.57 g, 57%).

¹H-NMR (500 MHz, CDCl₃) δ: 1.11 (3H, t, J=6.8 Hz), 1.25-1.41 (16H, m),1.59-1.67 (2H, m), 2.02-2.08 (4H, m), 2.77 (2H, t, J=6.6 Hz), 3.53 (2H,t, J=6.6 Hz), 4.06 (2H, s), 5.30-5.42 (4H, m), 9.74-9.75 (1H, m).

Reference Example 67(11Z,14Z)-1-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]icosa-11,14-dien-2-ol

To a solution of [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]acetaldehyde (1.27g, 4.1 mmol) obtained in Reference Example 66 in diethyl ether (12.4mL), 0.5 N linoleyl magnesium bromide (14 mL, 7.0 mmol) was added, andthe mixture was reacted at room temperature for 3 hours and subsequentlyat 60° C. for 3 hours. After treatment with a saturated aqueous solutionof ammonium chloride, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a yellow liquid (0.48 g, 21%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.23-1.46 (34H, m),1.52-1.61 (4H, m), 2.01-2.08 (8H, m), 2.31 (1H, d, J=3.1 Hz), 2.77 (4H,t, J=6.6 Hz), 3.23 (1H, dd, J=8.2, 9.4 Hz), 3.39-3.51 (3H, m), 3.72-3.81(1H, m), 5.29-5.43 (8H, m).

Example 47 3-(Dimethylamino)propyl(11Z,14Z)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icosa-11,14-dien-2-ylcarbonate

A solution of(11Z,14Z)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icosa-11,14-dien-2-ol(0.27 g, 0.48 mmol) obtained in Reference Example 67 and pyridine (0.24g, 3.0 mmol) in toluene (4.8 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.099 g, 0.33 mmol) in toluene (0.72 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 30 minutes,the reaction mixture was heated to room temperature, stirred for 1.5hours, and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.52 g,5.1 mmol) was added thereto, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (330 mg, 24%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88-0.94 (6H, m), 1.24-1.43 (34H, m),1.52-1.67 (4H, m), 1.86 (2H, tt, J=6.6, 7.4 Hz), 1.96-2.11 (8H, m), 2.24(6H, s), 2.38 (2H, t, J=7.4 Hz), 2.79 (4H, t, J=6.6 Hz), 3.38-3.54 (4H,m), 4.21 (2H, t, J=6.6 Hz), 4.82-4.89 (1H, m), 5.31-5.45 (8H, m).

MS (ESI+) m/z 688 [M+H]⁺

HRMS (ESI+) m/z 688.6269 (2.5 mDa).

Reference Example 6810-{2-[(2-Pentylcyclopropyl)methyl]cyclopropyl}-1-[(8-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}octyl)oxy]decan-2-ol

To a solution of a solution of 1.1 N diethylzinc in hexane (7.3 mL, 7.7mmol) in dichloromethane (12 mL) cooled to 0° C., chloroiodomethane (1.7g, 9.6 mmol) was added over 10 minutes, then a solution of(11Z,14Z)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icosa-11,14-dien-2-ol(0.27 g, 0.48 mmol) obtained in Reference Example 67 andchloroiodomethane (1.0 g, 5.7 mmol) in dichloromethane (7.0 mL) wasadded over 40 minutes, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution ofammonium chloride, the reaction mixture was subjected to extraction withdichloromethane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a yellow liquid (0.29 g, 98%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.32)-(−0.24) (4H, m), 0.57-0.84 (12H, m),0.85-0.92 (6H, m), 0.97-1.62 (50H, m), 2.31 (1H, d, J=3.1 Hz), 3.23 (1H,dd, J=8.2, 9.4 Hz), 3.39-3.52 (3H, m), 3.73-3.80 (1H, m).

Example 48 3-(Dimethylamino)propyl10-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}-1-[(8-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}octyl)oxy]decan-2-ylcarbonate

A solution of10-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}-1-[(8-{2-[(2-pentylcyclopropyl)methyl]cyclopropyl}octyl)oxy]decan-2-ol(0.17 g, 0.28 mmol) obtained in Reference Example 68 and pyridine (0.14g, 1.7 mmol) in toluene (2.8 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.056 g, 0.19 mmol) in toluene (0.42 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 1 hour, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 3-Dimethylamino-1-propanol (0.30 g, 2.9 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (135 mg, 66%).

¹H-NMR (400 MHz, CDCl₃) δ: (−0.34)-(−0.23) (4H, m), 0.57-0.84 (12H, m),0.86-0.92 (6H, m), 0.97-1.66 (50H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz),2.22 (6H, s), 2.36 (2H, t, J=7.4 Hz), 3.35-3.53 (4H, m), 4.18 (2H, t,J=6.6 Hz), 4.80-4.87 (1H, m).

MS (ESI+) m/z 744 [M+H]⁺

HRMS (ESI+) m/z 744.6876 (0.6 mDa).

Reference Example 69 1-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]icosan-2-ol

To a solution of [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]acetaldehyde (0.22g, 0.71 mmol) obtained in Reference Example 66 in tetrahydrofuran (2.9mL), 0.5 N octadecyl magnesium chloride (3.0 mL, 1.5 mmol) was added,and the mixture was reacted overnight at room temperature. Aftertreatment with a saturated aqueous solution of ammonium chloride, thereaction mixture was subjected to extraction with ethyl acetate, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a yellow liquid (0.13 g, 31%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.20-1.62 (52H, m),2.01-2.08 (4H, m), 2.31 (1H, d, J=3.1 Hz), 2.77 (2H, t, J=6.6 Hz), 3.23(1H, dd, J=8.2, 9.4 Hz), 3.39-3.55 (3H, m), 3.73-3.80 (1H, m), 5.29-5.42(4H, m).

Example 49 3-(Dimethylaminopropyl)1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icosan-2-yl carbonate

A solution of 1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icosan-2-ol (0.13g, 0.22 mmol) obtained in Reference Example 69 and pyridine (0.11 g, 1.4mmol) in toluene (2.2 mL) was cooled to 0° C. in an ice bath, and asolution of triphosgene (0.045 g, 0.15 mmol) in toluene (0.33 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 40 minutes,the reaction mixture was heated to room temperature, stirred for 1 hour,and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.24 g, 2.3 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (86 mg, 56%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.41 (48H, m),1.50-1.66 (4H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 2.01-2.09 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.4 Hz), 2.77 (2H, t, J=6.6 Hz), 3.35-3.53 (4H,m), 4.18 (2H, t, J=6.6 Hz), 4.80-4.86 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 692 [M+H]⁺

HRMS (ESI+) m/z 692.6588 (3.1 mDa).

Reference Example 70 (11Z,14Z)-1-(Octadecyloxy)icosa-11,14-dien-2-ol

To a solution of octadecan-1-yloxyacetaldehyde (0.25 g, 0.80 mmol) intetrahydrofuran (2.4 mL), 0.5 N linoleyl magnesium bromide (2.4 mL, 1.2mmol) was added, and the mixture was reacted at room temperature for 4hours. After treatment with a saturated aqueous solution of ammoniumchloride, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a yellow liquid (0.17 g, 38%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88-0.94 (6H, m), 1.22-1.70 (52H, m),2.04-2.11 (4H, m), 2.35 (1H, d, J=3.1 Hz), 2.80 (2H, t, J=6.6 Hz), 3.26(1H, dd, J=8.2, 9.4 Hz), 3.42-3.58 (3H, m), 3.75-3.83 (1H, m), 5.32-5.45(4H, m).

Example 50 3-(Dimethylaminopropyl)(11Z,14Z)-1-(octadecyloxy)icosa-11,14-dien-2-yl carbonate

A solution of (11Z,14Z)-1-(octadecyloxy)icosa-11,14-dien-2-ol (0.17 g,0.30 mmol) obtained in Reference Example 70 and pyridine (0.15 g, 1.9mmol) in toluene (3.0 mL) was cooled to 0° C. in an ice bath, and asolution of triphosgene (0.062 g, 0.21 mmol) in toluene (0.45 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 30 minutes, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 3-Dimethylamino-1-propanol (0.33 g, 3.2 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (53 mg, 25%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.21-1.40 (48H, m),1.50-1.65 (4H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 2.01-2.09 (4H, m), 2.22(6H, s), 2.36 (2H, t, J=7.4 Hz), 2.77 (2H, t, J=6.6 Hz), 3.35-3.53 (4H,m), 4.18 (2H, t, J=6.6 Hz), 4.80-4.87 (1H, m), 5.29-5.42 (4H, m).

MS (ESI+) m/z 692 [M+H]⁺

HRMS (ESI+) m/z 692.6578 (2.1 mDa).

Reference Example 71(11Z,14R)-1-(Octadecyloxy)-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ol

Dried magnesium (shavings, 0.48 g, 19.8 mmol) was dipped intetrahydrofuran (4.0 mL). 1,2-Dibromoethane (3 drops) was added thereto,and the mixture was vigorously stirred. After the solution turnedblack-gray, a solution of2-{[(7R,9Z)-18-bromooctadec-9-en-7-yl]oxy}tetrahydro-2H-pyran (5.70 g,13.2 mmol) obtained in Reference Example 53 in tetrahydrofuran (18.5 mL)was added thereto over 40 minutes, and the mixture was stirred at roomtemperature for 3 hours.

A 7.5 mL aliquot of the obtained Grignard reagent solution was added toa solution of octadecan-1-yloxyacetaldehyde (0.78 g, 2.5 mmol) intetrahydrofuran (7.5 mL), and the mixture was reacted at roomtemperature for 6 hours. After treatment with a saturated aqueoussolution of ammonium chloride, the reaction mixture was subjected toextraction with ethyl acetate, and the obtained organic layer was driedover anhydrous magnesium sulfate. The solvent was distilled off underreduced pressure, and the residue was then subjected to silica gelcolumn chromatography to obtain the compound of interest as a colorlessliquid (0.61 g, 37%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.89 (6H, t, J=7.0 Hz), 1.20-2.40 (64H, m),3.23 (1H, dd, J=8.2, 9.4 Hz), 3.39-3.56 (5H, m), 3.58-3.70 (1H, m),3.73-3.81 (1H, m), 3.87-3.98 (1H, m), 4.64-4.74 (1H, m), 5.34-5.50 (2H,m).

Reference Example 72 3-(Dimethylamino)propyl(11Z,14R)-1-(octadecyloxy)-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ylcarbonate

A solution of(11Z,14R)-1-(octadecyloxy)-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ol(0.61 g, 0.92 mmol) obtained in Reference Example 71 and pyridine (0.46g, 5.8 mmol) in toluene (9.2 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.19 g, 0.63 mmol) in toluene (1.4 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 15 minutes, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 3-Dimethylamino-1-propanol (0.99 g, 9.6 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.41 g, 56%).

Reference Example 73 3-(Dimethylamino)propyl(11Z,14R)-14-hydroxy-1-(octadecyloxy)icos-11-en-2-yl carbonate

To a solution of 3-(dimethylamino)propyl(11Z,14R)-1-(octadecyloxy)-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ylcarbonate (0.41 g, 0.52 mmol) obtained in Reference Example 72 inethanol (5.2 mL), a 2 N aqueous hydrochloric acid solution (2.6 mL, 5.2mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction with ethylacetate, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.34 g, 92%).

Example 51(7R,9Z)-19-({[3-(Dimethylamino)propoxy]carbonyl}oxy)-20-(octadecyloxy)icos-9-en-7-ylacetate

To a solution of 3-(dimethylamino)propyl(11Z,14R)-14-hydroxy-1-(octadecyloxy)icos-11-en-2-yl carbonate (0.11 g,0.16 mmol) obtained in Reference Example 73 and pyridine (0.25 g, 3.1mmol) in dichloromethane (1.6 mL), acetic acid chloride (0.12 g, 1.6mmol) was added, and the mixture was reacted at room temperature for 3hours. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (75 mg, 64%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.21-1.37 (50H, m),1.50-1.66 (6H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.97-2.06 (5H, m), 2.22(6H, s), 2.25-2.31 (2H, m), 2.36 (2H, t, J=7.4 Hz), 3.35-3.51 (4H, m),4.18 (2H, t, J=6.6 Hz), 4.79-4.91 (2H, m), 5.28-5.36 (1H, m), 5.43-5.51(1H, m).

MS (ESI+) m/z 752 [M+H]⁺

HRMS (ESI+) m/z 752.6775 (3.4 mDa).

Reference Example 74(11Z,14R)-1-[(9Z,12Z)-Octadeca-9,12-dien-1-yloxy]-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ol

Dried magnesium (shavings, 0.24 g, 9.7 mmol) was dipped intetrahydrofuran (3.0 mL). 1,2-Dibromoethane (4 drops) was added thereto,and the mixture was vigorously stirred. After the solution turnedblack-gray, a solution of2-{[(7R,9Z)-18-bromooctadec-9-en-7-yl]oxy}tetrahydro-2H-pyran (2.8 g,6.5 mmol) obtained in Reference Example 53 in tetrahydrofuran (18.5 mL)was added thereto over 40 minutes, and the mixture was stirred overnightat room temperature.

To the obtained Grignard reagent solution,[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]acetaldehyde (2.0 g, 6.5 mmol)obtained in Reference Example 66 was added, and the mixture was reactedat 60° C. for 2 hours. After treatment with a saturated aqueous solutionof ammonium chloride, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.27 g, 6%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.22-2.40 (54H, m), 2.77(2H, t, J=6.6 Hz), 3.23 (1H, dd, J=8.2, 9.4 Hz), 3.39-3.56 (5H, m),3.58-3.72 (1H, m), 3.72-3.81 (1H, m), 3.86-3.98 (1H, m), 4.64-4.74 (1H,m), 5.28-5.49 (6H, m).

Reference Example 75 3-(Dimethylamino)propyl(11Z,14R)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ylcarbonate

A solution of(11Z,14R)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ol(0.27 g, 0.41 mmol) obtained in Reference Example 74 and pyridine (0.20g, 2.6 mmol) in toluene (4.1 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.084 g, 0.28 mmol) in toluene (0.61 mL) wasadded thereto over 2 minutes. After stirring at 0° C. for 15 minutes,the reaction mixture was heated to room temperature, stirred for 1 hour,and cooled to 0° C. again. 3-Dimethylamino-1-propanol (0.44 g, 4.3 mmol)was added thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.16 g, 48%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.23-1.88 (50H, m),1.98-2.09 (8H, m), 2.22 (6H, m), 2.36 (2H, t, J=7.4 Hz), 2.77 (2H, t,J=6.6 Hz), 3.35-3.52 (5H, m), 3.59-3.71 (1H, m), 3.87-3.97 (1H, m), 4.18(2H, t, J=6.6 Hz), 4.63-4.73 (1H, m), 4.80-4.87 (1H, m), 5.29-5.49 (6H,m).

Reference Example 76 3-(Dimethylamino)propyl(11Z,14R)-14-hydroxy-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icos-11-en-2-ylcarbonate

To a solution of 3-(dimethylamino)propyl(11Z,14R)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-14-(tetrahydro-2H-pyran-2-yloxy)icos-11-en-2-ylcarbonate (0.16 g, 0.20 mmol) obtained in Reference Example 75 inethanol (0.98 mL), a 2 N aqueous hydrochloric acid solution (0.49 mL,0.98 mmol) was added, and the mixture was reacted at room temperaturefor 3 hours. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.08 g, 60%).

Example 52(7R,9Z)-19-({[3-(Dimethylamino)propoxy]carbonyl}oxy)-20-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icos-9-en-7-ylacetate

To a solution of 3-(dimethylamino)propyl(11Z,14R)-14-hydroxy-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]icos-11-en-2-ylcarbonate (0.08 g, 0.11 mmol) obtained in Reference Example 76 andpyridine (0.37 g, 4.5 mmol) in dichloromethane (2.3 mL), acetyl chloride(0.18 g, 2.3 mmol) was added, and the mixture was reacted at roomtemperature for 3 hours. After treatment with a saturated aqueoussolution of sodium bicarbonate, the reaction mixture was subjected toextraction with hexane, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(53 mg, 63%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.23-1.40 (36H, m),1.50-1.64 (6H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.97-2.09 (9H, m), 2.22(6H, s), 2.25-2.32 (2H, m), 2.36 (2H, t, J=7.4 Hz), 2.77 (2H, t, J=6.6Hz), 3.35-3.52 (4H, m), 4.18 (2H, t, J=6.6 Hz), 4.80-4.91 (2H, m),5.28-5.51 (6H, m).

MS (ESI+) m/z 748 [M+H]⁺

HRMS (ESI+) m/z 748.6489 (3.4 mDa).

Reference Example 772-{[(9Z,12R)-12-(Tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}ethanol

To a suspension of sodium hydride (64%, 2.97 g, 79.2 mmol) inN,N-dimethylformamide, ethylene glycol (5.25 g, 84.5 mmol) was added,and the mixture was stirred at 80° C. for 45 minutes.(9Z,12R)-12-Hydroxyoctadec-9-en-1-yl methanesulfonate (11.8 g, 26.4mmol) obtained in Reference Example 51 was added thereto, and themixture was reacted at 80° C. for 2 hours. After treatment with water,the reaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (8.36 g, 77%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 1.23-1.62 (26H, m),1.66-1.75 (1H, m), 1.78-1.87 (1H, m), 1.97-2.07 (3H, m), 2.23 (1H, t,J=6.3 Hz), 2.26-2.41 (1H, m), 3.47 (2H, t, J=6.6 Hz), 3.53 (2H, dd,J=3.1, 5.1 Hz), 3.58-3.72 (1H, m), 3.87-3.98 (1H, m), 4.64-4.74 (1H, m),5.33-5.49 (2H, m).

Reference Example 78{[(9Z,12R)-12-(Tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}acetaldehyde

To a solution of2-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}ethanol(5.36 g, 13.0 mmol) obtained in Reference Example 77 and triethylamine(6.57 g, 65.0 mmol) in dimethyl sulfoxide (26.0 mL), a sulfurtrioxide-pyridine complex (5.17 g, 32.5 mmol) was added, and the mixturewas reacted at room temperature for 2.5 hours. The reaction mixture wasdiluted with water and then subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a yellow liquid (3.96 g, 74%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 1.23-1.67 (26H, m),1.66-1.75 (1H, m), 1.78-1.86 (1H, m), 1.98-2.07 (3H, m), 2.20-2.41 (2H,m), 3.44-3.54 (3H, m), 3.55-3.71 (1H, m), 3.87-3.97 (1H, m), 4.06 (2H,s), 4.64-4.74 (1H, m), 5.32-5.49 (2H, m), 9.75 (1H, s).

Reference Example 791-{[(9Z,12R)-12-(Tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosan-2-ol

To a solution of{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}acetaldehyde(0.40 g, 0.97 mmol) obtained in Reference Example 78 in tetrahydrofuran(2.9 mL), 0.5 N octadecyl magnesium chloride (2.4 mL, 1.2 mmol) wasadded, and the mixture was reacted overnight at room temperature. Aftertreatment with a saturated aqueous solution of ammonium chloride, thereaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a yellow liquid (0.24 g, 37%).

Reference Example 80 3-(Dimethylamino)propyl1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosan-2-ylcarbonate

A solution of1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosan-2-ol(0.24 g, 0.36 mmol) obtained in Reference Example 79 and pyridine (0.18g, 2.3 mmol) in toluene (3.6 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.074 g, 0.25 mmol) in toluene (0.54 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 10 minutes, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 3-Dimethylamino-1-propanol (0.39 g, 3.8 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.24 g, 84%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, t, J=7.0 Hz), 1.21-1.75 (60H, m),1.81-2.40 (8H, m), 2.22 (6H, s), 3.35-3.53 (5H, m), 3.58-3.72 (1H, m),3.87-3.97 (1H, m), 4.18 (2H, t, J=6.6 Hz), 4.64-4.73 (1H, m), 4.80-4.87(1H, m), 5.32-5.49 (2H, m).

Reference Example 81 3-(Dimethylamino)propyl1-{[(9Z,12R)-12-hydroxyoctadec-9-en-1-yl]oxy}icosan-2-yl carbonate

To a solution of 3-(dimethylamino)propyl1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosan-2-ylcarbonate (0.24 g, 0.30 mmol) obtained in Reference Example 80 inethanol (3.0 mL), a 2 N aqueous hydrochloric acid solution (1.5 mL, 3.0mmol) was added, and the mixture was reacted at room temperature for 2hours. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.16 g, 76%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.21-1.65 (54H, m), 1.84(2H, dd, J=6.6, 7.4 Hz), 2.05 (2H, q, J=6.6 Hz), 2.18-2.24 (2H, m), 2.22(6H, s), 2.36 (2H, t, J=7.4 Hz), 3.35-3.52 (4H, m), 3.37-3.65 (1H, m),4.18 (2H, t, J=6.6 Hz), 4.83 (1H, quint, J=5.9 Hz), 5.35-5.44 (1H, m),5.52-5.61 (1H, m).

Example 53(20,23R)-2-Methyl-9-octadecyl-7-oxo-6,8,11-trioxa-2-azanonacos-20-en-23-ylacetate

To a solution of 3-(dimethylamino)propyl1-{[(9Z,12R)-12-hydroxyoctadec-9-en-1-yl]oxy}icosan-2-yl carbonate (0.16g, 0.23 mmol) obtained in Reference Example 81 and pyridine (0.36 g, 4.6mmol) in dichloromethane (2.3 mL), acetyl chloride (0.18 g, 2.3 mmol)was added, and the mixture was reacted overnight at room temperature.After treatment with a saturated aqueous solution of sodium bicarbonate,the reaction mixture was subjected to extraction with hexane, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a colorless liquid (121 mg, 70%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.91 (6H, m), 1.22-1.38 (50H, m),1.49-1.65 (6H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.97-2.06 (5H, m), 2.22(6H, s), 2.25-2.32 (2H, m), 2.36 (2H, t, J=7.4 Hz), 3.35-3.53 (4H, m),4.18 (2H, t, J=6.6 Hz), 4.79-4.91 (2H, m), 5.28-5.36 (1H, m), 5.43-5.51(1H, m).

MS (ESI+) m/z 752 [M+H]⁺

HRMS (ESI+) m/z 752.6802 (3.4 mDa).

Reference Example 82(11Z,14Z)-1-{[(9Z,12R)-12-(Tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ol

To a solution of{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}acetaldehyde(0.40 g, 0.97 mmol) obtained in Reference Example 78 in tetrahydrofuran(2.9 mL), 0.5 N linoleyl magnesium bromide (2.9 mL, 1.5 mmol) was added,and the mixture was reacted overnight at room temperature. Aftertreatment with a saturated aqueous solution of ammonium chloride, thereaction mixture was subjected to extraction with ethyl acetate, and theobtained organic layer was dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure, and the residue wasthen subjected to silica gel column chromatography to obtain thecompound of interest as a yellow liquid (0.37 g, 57%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.23-1.87 (48H, m),1.98-2.40 (9H, m), 2.77 (2H, t, J=6.6 Hz), 3.23 (1H, dd, J=8.2, 9.4 Hz),3.39-3.52 (4H, m), 3.58-3.71 (1H, m), 3.73-3.80 (1H, m), 3.87-3.97 (1H,m), 4.64-4.74 (1H, m), 5.29-5.49 (6H, m).

Reference Example 83 3-(Dimethylamino)propyl(11Z,14Z)-1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ylcarbonate

A solution of(11Z,14Z)-1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ol(0.37 g, 0.56 mmol) obtained in Reference Example 82 and pyridine (0.28g, 3.5 mmol) in toluene (5.6 mL) was cooled to 0° C. in an ice bath, anda solution of triphosgene (0.11 g, 0.39 mmol) in toluene (0.84 mL) wasadded thereto over 1 minute. After stirring at 0° C. for 30 minutes, thereaction mixture was heated to room temperature, stirred for 1 hour, andcooled to 0° C. again. 3-Dimethylamino-1-propanol (0.61 g, 5.9 mmol) wasadded thereto, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.33 g, 75%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.23-1.75 (48H, m), 1.84(2H, tt, J=6.6, 7.4 Hz), 1.98-2.40 (8H, m), 2.22 (6H, s), 2.36 (2H, t,J=7.4 Hz), 2.77 (2H, t, J=6.3 Hz), 3.35-3.53 (5H, m), 3.58-3.71 (1H, m),3.87-3.97 (1H, m), 4.18 (2H, t, J=6.6 Hz), 4.64-4.74 (1H, m), 4.80-4.87(1H, m), 5.29-5.48 (6H, m).

Reference Example 84 3-(Dimethylamino)propyl(11Z,14Z)-1-{[(9Z,12R)-12-hydroxyoctadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ylcarbonate

To a solution of 3-(dimethylamino)propyl(11Z,14Z)-1-{[(9Z,12R)-12-(tetrahydro-2H-pyran-2-yloxy)octadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ylcarbonate (0.33 g, 0.42 mmol) obtained in Reference Example 83 inethanol (4.2 mL), a 2 N aqueous hydrochloric acid solution (2.1 mL, 4.2mmol) was added, and the mixture was reacted overnight at roomtemperature. After treatment with a saturated aqueous solution of sodiumbicarbonate, the reaction mixture was subjected to extraction withhexane-ethyl acetate, and the obtained organic layer was dried overanhydrous magnesium sulfate. The solvent was distilled off under reducedpressure, and the residue was then subjected to silica gel columnchromatography to obtain the compound of interest as a colorless liquid(0.29 g, 98%).

Example 54(20,23R)-2-Methyl-9-[(9Z,12Z)-octadeca-9,12-dien-1-yl]-7-oxo-6,8,11-trioxa-2-azanonacos-20-en-23-ylacetate

To a solution of 3-(dimethylamino)propyl(11Z,14Z)-1-{[(9Z,12R)-12-hydroxyoctadec-9-en-1-yl]oxy}icosa-11,14-dien-2-ylcarbonate (0.51 g, 0.72 mmol) obtained in Reference Example 84 andpyridine (1.1 g, 14 mmol) in dichloromethane (7.2 mL), acetyl chloride(0.57 g, 7.2 mmol) was added, and the mixture was reacted overnight atroom temperature. After treatment with a saturated aqueous solution ofsodium bicarbonate, the reaction mixture was subjected to extractionwith hexane, and the obtained organic layer was dried over anhydrousmagnesium sulfate. The solvent was distilled off under reduced pressure,and the residue was then subjected to silica gel column chromatographyto obtain the compound of interest as a colorless liquid (0.42 g, 78%).

¹H-NMR (400 MHz, CDCl₃) δ: 0.85-0.92 (6H, m), 1.23-1.40 (36H, m),1.49-1.65 (6H, m), 1.84 (2H, tt, J=6.6, 7.4 Hz), 1.98-2.08 (9H, m), 2.22(6H, s), 2.25-2.32 (2H, m), 2.36 (2H, t, J=7.4 Hz), 2.77 (2H, t, J=6.6Hz), 3.35-3.53 (4H, m), 4.18 (2H, t, J=6.6 Hz), 4.80-4.91 (2H, m),5.28-5.51 (6H, m).

MS (ESI+) m/z 748 [M+H]⁺

HRMS (ESI+) m/z 748.6476 (2.1 mDa).

Example 55 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

A dispersion of a nucleic acid lipid particle containing the compounddescribed in Reference Example 2, Example 41, Example 42, or Example 43was obtained in the same way as in Example 31. The obtained nucleic acidlipid particle-containing dispersion was characterized by the followingmethods.

(1) Average Particle Size

The particle size of the liposome was measured using ZetaPotential/Particle Sizer NICOMP™ 380ZLS (Particle Sizing Systems, LLC).In the tables, the average particle size is indicated by avolume-average particle size, and the numeric value following ±represents a deviation.

(2) Rate of Encapsulation of Double-stranded Polynucleotide

The rate of encapsulation of the double-stranded polynucleotide wasmeasured using Quant-iT RiboGreen RNA Assay kit (Invitrogen Corp.)according to the attached document.

Specifically, the double-stranded polynucleotide in the nucleic acidlipid particle dispersion was quantified in the presence and absence ofa 0.015% Triton X-100 detergent, and the rate of encapsulation wascalculated according to the following expression:{[Amount of the double-stranded polynucleotide in the presence of thedetergent]−[Amount of the double-stranded polynucleotide in the absenceof the detergent]}/[Amount of the double-stranded polynucleotide in thepresence of the detergent]}×100 (%)(3) Ratio of Double-stranded Polynucleotide to Lipid

The amount of the double-stranded polynucleotide in a sample from thenucleic acid lipid particle dispersion was measured in the presence of a5% Triton X-100 detergent by ion-exchange chromatography (system:Agilent 1100 series, column: DNAPac PA200 (4×250 mm) (Thermo FisherScientific K.K.), buffer A: 10 mM Tris, 25 mM sodium perchlorate, and20% ethanol, pH 7.0, buffer B: 10 mM Tris, 250 mM sodium perchlorate,and 20% ethanol, pH 7.0, gradient (B%): 20-70% (0-15 min), flow rate:0.5 mL/min, temperature: 40° C., detection: 260 nm).

The amount of the phospholipid in the nucleic acid lipid particledispersion was measured using Phospholipid C-Test Wako (Wako PureChemical Industries Ltd.) according to the attached document.Specifically, the phospholipid in the sample was quantified in thepresence of a 1% Triton X-100 detergent.

The amounts of cholesterol and LP in the nucleic acid lipid particledispersion were measured by reverse-phase chromatography (system:Agilent 1100 series, column: Chromolith Performance RP-18 endcapped100-3 monolithic HPLC-column (Merck), buffer A: 0.01% trifluoroaceticacid, buffer B: 0.01% trifluoroacetic acid and methanol, gradient (B %):82-92% (0-10 min), flow rate: 2 mL/min, temperature: 50° C., detection:205 nm).

The total amount of lipids was calculated from the amount of thephospholipid, the amount of cholesterol, and the amount of LP, and thecompositional ratio of lipid components constituting the liposome, andthe ratio of the polynucleotide to the lipid was calculated from theaforementioned amount of the polynucleotide and the total amount oflipids according to the following expression:[Double-stranded polynucleotide concentration]/[Total lipidconcentration] (wt/wt)

The results are shown in Table 16.

TABLE 16 Ratio of Rate of polynucleotide to Average polynucleotide lipidparticle size LP name encapsulation (%) siRNA/lipid (wt/wt) (nm)Reference 94 0.055 165 ± 11 Example 2 Example 41 97 0.101 158 ± 40Example 42 97 0.077 188 ± 45 Example 43 97 0.075 174 ± 31

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 100 nm to approximately200 nm.

Test Example 8

By the same procedures as in Test Example 3, SW480 cells were treatedwith 50 nM, 5 nM, 0.5 nM, and 0.05 nM each of nucleic acid lipidparticles each prepared using a novel lipid, and the strength of humanβ-catenin gene expression inhibitory activity was compared among theseparticles.

As a result, as shown in Table 17, the nucleic acid lipid particlecontaining the compound of Example 41, 42, or 43 exhibited stronginhibitory activity against β-catenin gene expression, as compared withthe nucleic acid lipid particle containing the lipid of ReferenceExample 2 used as a control. These results demonstrated that thecompound of Example 8 is a novel lipid useful for preparing nucleic acidlipid particles that exhibit strong activity.

TABLE 17 β-catenin gene expression inhibitory activity IC50 (nM)Reference Example 2 >50 Example 41 2.6 Example 42 6.0 Example 43 24

Example 56 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

A lipid solution having a total lipid concentration of 26.8 mM inethanol with distearoylphosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter, referred to asDSPC, NOF CORPORATION), cholesterol (hereinafter, referred to as Chol,Sigma-Aldrich, Inc.), the compound described in Example 23 or 28(hereinafter, referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DMA) were prepared at a molar ratio ofDSPC:Chol:LP:PEG-C-DMA=10:48:40:2.

The concentration of a double-stranded polynucleotide described inNature Biotechnology (2008) 26, 561-569 (siFVII: siRNA against mouseFactor VII) was adjusted to 1 mg/mL with a citrate buffer solution (10mM citrate buffer, pH 4.0) containing 30% ethanol to obtain adouble-stranded polynucleotide solution.

The lipid solution, the double-stranded polynucleotide solution, and acitrate buffer solution (20 mM citrate buffer, pH 4.0) were heated to37° C. The lipid solution was added dropwise to the citrate buffersolution (20 mM citrate buffer, pH 4.0) and mixed therewith such thatthe volume ratio between the lipid solution and the citrate buffersolution was 3:7 to obtain a crude liposome dispersion. Subsequently,the crude liposome dispersion was added dropwise to the double-strandedpolynucleotide solution and mixed therewith such that the ratio (N/P) ofLP-derived nitrogen atoms (N) to double-stranded polynucleotide-derivedphosphorus atoms (P) was 3. The mixture was incubated at 37° C. for 30minutes to obtain a nucleic acid lipid particle dispersion. The nucleicacid lipid particle dispersion was dialyzed against approximately 100 mLof a phosphate buffer solution (pH 7.4) for 12 to 18 hours(Float-A-Lyzer G2, MWCO: 100 kD, Spectra/Por) for the removal of ethanoland the removal of unencapsulated double-stranded polynucleotides byneutralization to obtain a purified dispersion of a nucleic acid lipidparticle containing the double-stranded polynucleotide and the compounddescribed in Example 23 or 28.

The obtained nucleic acid lipid particle was characterized by themethods described in Example 55, and the rate of polynucleotideencapsulation in the nucleic acid lipid particle, the weight ratio ofthe polynucleotide to the lipid, and the average particle size are shownin Table 18.

TABLE 18 Ratio of Rate of polynucleotide to Average polynucleotide lipidparticle size LP name encapsulation (%) siRNA/lipid (wt/wt) (nm) Example23 99 0.076 219 ± 49 Example 28 97 0.083 177 ± 36

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 100 nm to approximately300 nm.

Test Example 9 Factor VII (FVII) Protein Measurement

By the same procedures as in Test Example 7, the strength of Factor VIIprotein expression inhibitory activity was compared among nucleic acidlipid particles each prepared using a novel lipid. The prepared nucleicacid lipid particle dispersion was intravenously injected at a dose of0.1 mg/kg to the tail of each mouse. One day and 6 days after theadministration, approximately 50 μL of blood was collected from the tailvein, and plasma was obtained.

The results obtained 1 day and 6 days after the administration are eachshown in Table 19. As a result, the nucleic acid lipid particlecontaining the compound of Example 23 or 28 exhibited strong FVIIinhibitory activity. These results demonstrated that the nucleic acidlipid particle having the compound of Example 23 or 28 is useful as anucleic acid lipid particle capable of inhibiting gene expression.

TABLE 19 Relative amount of FVII (%) 1 day later 6 days later PBS 100100 Example 23 21 32 Example 28 45 57

Example 57 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

A lipid solution having a total lipid concentration of 6.5 mM in ethanolwith dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine: hereinafter, referred toas DPPC, NOF CORPORATION), cholesterol (hereinafter, referred to asChol, Sigma-Aldrich, Inc.), the compound described in Example 19, 45, or54 (hereinafter, referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearoyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DSA) were prepared at a molar ratio ofDPPC:Chol:LP:PEG-C-DSA=7:33.5:57:2.5.

The concentration of a double-stranded polynucleotide described inJournal Clinical Investigation (2009) 119, 661-673 (PLK1424-2/A: siRNAagainst mouse PLK1) was adjusted to 1 mg/mL with a citrate buffersolution (10 mM citrate buffer, pH 4.0) containing 30% ethanol to obtaina double-stranded polynucleotide solution.

The lipid solution, the double-stranded polynucleotide solution, and acitrate buffer solution (20 mM citrate buffer, pH 4.0) were heated to37° C. The lipid solution was added dropwise to the citrate buffersolution (20 mM citrate buffer, pH 4.0) and mixed therewith such thatthe volume ratio between the lipid solution and the citrate buffersolution was 3:7 to obtain a crude liposome dispersion. Subsequently,the crude liposome dispersion was added dropwise to the double-strandedpolynucleotide solution and mixed therewith such that the ratio (N/P) ofLP-derived nitrogen atoms (N) to double-stranded polynucleotide-derivedphosphorus atoms (P) was 3. The mixture was incubated at 37° C. for 30minutes to obtain a nucleic acid lipid particle dispersion. The nucleicacid lipid particle dispersion was dialyzed against approximately 100 mLof a phosphate buffer solution (pH 7.4) for 12 to 18 hours(Float-A-Lyzer G2, MWCO: 100 kD, Spectra/Por) for the removal of ethanoland the removal of unencapsulated double-stranded polynucleotides byneutralization to obtain a purified dispersion of a nucleic acid lipidparticle containing the double-stranded polynucleotide and the compounddescribed in Example 19, 45, or 54.

The obtained nucleic acid lipid particle was characterized by themethods described in Example 55, and the rate of polynucleotideencapsulation in the nucleic acid lipid particle, the weight ratio ofthe polynucleotide to the lipid, and the average particle size are shownin Table 20.

TABLE 20 Ratio of Rate of polynucleotide to Average polynucleotide lipidparticle size LP name encapsulation (%) siRNA/lipid (wt/wt) (nm) Example19 97 0.095 103 ± 16 Example 45 96 0.093 100 ± 27 Example 54 98 0.078127 ± 29

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 50 nm to approximately 150nm.

Test Example 10

After acclimatization and raising of each nude mouse(CAnN.Cg-Foxn1[nu]/Crl Crlj[Foxn1nu/Foxn1nu]) for 8 days, cultured humanHep3B cells (1×10⁷ cells/mouse) were subcutaneously transplanted to theright lateral region of the mouse. 19 days after the tumortransplantation, the mice were grouped with the tumor volume as anindex, and, on the next day, the nucleic acid lipid particle-containingdispersion prepared in Example 57 was intravenously administered(administered at doses of 1 mg/kg and 3 mg/kg) to the tail of eachmouse. PBS was administered to a control group. On the day after theadministration of the nucleic acid lipid particle, a tumor mass wascollected from the cancer-bearing mouse, and a nucleic acid wasextracted using QIAzol Lysis Reagent (manufactured by Qiagen N.V.) andchloroform. Then, total RNA was purified using QIAGNE RNeasy Plus Minikit (manufactured by Qiagen N.V.) according to the attached protocol.

16 μL of the purified RNA and 4 μL of SuperScript VILO Master mix (LifeTechnologies, Inc.) were mixed and used in RT reaction under conditionsgiven below.

RT reaction: 25° C. for 10 min

-   -   42° C. for 60 min    -   85° C. for 5 min.

The probes for real-time PCR used were TaqMan(R) Gene Expression Assays(PLK-1, FAM probe, Hs00153444_m1, manufactured by Applied Biosystems,Inc.) as a human PLK-1 gene probe and TaqMan(R) Gene Expression Assays(VIC probe, Hs99999905_m1, manufactured by Applied Biosystems, Inc.) asa human GAPDH gene probe as an internal standard. 5 μL of TaqMan(R) FastAdvanced Master Mix, 2.66 μL of RNase-Free Water, 0.17 μL of each geneprobe, and 2 μL of the prepared cDNA solution were added per well of a384-well PCR plate (manufactured by Applied Biosystems, Inc.) to bringthe total amount to 10 μL, which was then loaded in ViiA™ 7 Real-timePCR system (manufactured by Applied Biosystems, Inc.) and subjected toPCR under conditions given below. The real-time PCR was carried out atN=4 for the prepared cDNA.

PCR initial activation: 95° C. for 20 seconds

PCR: 95° C. for 1 second

-   -   62° C. for 20 seconds

This PCR cycle was repetitively performed 40 times.

The quantitative analysis was conducted by the ΔΔCt method. A value(ΔΔCt) was determined by subtracting ΔCt of the PBS administration groupfrom the difference in Ct value (ΔCt) between human PLK-1 and humanGAPDH in the administration group of each nucleic acid lipid particle,and a relative value (RQ) to the PBS administration group, RQmax, andRQmin were calculated according to the following expressions:RQ=2^(−ΔΔCt)RQmax=2^(−95% CI of ΔΔCt) (95% CI of ΔΔCt: the maximum value of 95%confidence interval of ΔΔCt)RQmin=2^(−95% CI of ΔΔCt) (95% CI of ΔΔCt: the minimum value of 95%confidence interval of ΔΔCt)

(CI: Confidence Interval)

The results are shown in FIG. 5. In the diagram, error bars werecalculated according to the following expression:+error bars: RQmax−RQ, −error bars: RQ−RQmin)

As a result, as shown in FIG. 5, the nucleic acid lipid particle havingthe compound of Example 19, 45, or 54 exhibited strong PLK-1 expressioninhibitory activity in tumor. These results demonstrated that thenucleic acid lipid particle having the compound of Example 19, 45, or 54is useful as a nucleic acid lipid particle capable of inhibiting geneexpression.

Example 58 Preparation of mRNA-encapsulated Nucleic Acid Lipid Particle

Distearoylphosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter, referred to asDSPC, NOF CORPORATION), cholesterol (hereinafter, referred to as Chol,Sigma-Aldrich, Inc.), the compound described in Example 8 (hereinafter,referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DMA) were dissolved at a molar ratio ofDSPC:Chol:LP:PEG-C-DMA=10:48:40:2 in ethanol to give a total lipidconcentration of 10 mM. The obtained lipid solution was added dropwiseto a citrate buffer solution (20 mM citrate buffer, pH 4.0) and mixedtherewith such that the volume ratio between the lipid solution and thecitrate buffer solution was 3:7 to obtain a crude dispersion of a lipidparticle.

On the other hand, the concentration of mCherry mRNA (5meC, ψ)(hereinafter, referred to as mCherry mRNA, Catalog No.: L-6113, TriLinkBioTechnologies, Inc.) or firefly luciferase mRNA (5meC, ψ)(hereinafter, referred to as FLuc mRNA, Catalog No.: L-6107, TriLinkBioTechnologies, Inc.) was adjusted to 0.1 mg/mL with a citrate buffersolution (20 mM citrate buffer, pH 4.0) containing 30% ethanol.

Subsequently, 790 μL of the crude lipid particle dispersion and 350 μLof the mRNA solution were mixed such that the ratio (N/P) of the numberof LP molecules (N) to the number of mRNA-derived phosphorus atoms (P)was N/P molar ratio=9.0. The mixture was incubated at 37° C. for 30minutes to obtain a nucleic acid lipid particle dispersion. The nucleicacid lipid particle dispersion was dialyzed against approximately 100 mLof a phosphate buffer solution (pH 7.4) for 12 to 18 hours(Float-A-Lyzer G2, MWCO: 100 kD, Spectra/Por) for the removal of ethanolto obtain a purified dispersion of an mRNA-encapsulated nucleic acidlipid particle containing the compound described in Example 8.

Example 59 Characterization of mRNA-encapsulated Nucleic Acid LipidParticle

The nucleic acid lipid particle-containing dispersion prepared inExample 58 was characterized. Each characterization method will bedescribed.

(1) Average Particle Size

The particle size of the liposome was measured using ZetaPotential/Particle Sizer NICOMP™ 380ZLS (Particle Sizing Systems, LLC).In the tables, the average particle size is indicated by avolume-average particle size, and the numeric value following ±represents a deviation.

(2) Rate of Encapsulation of mRNA

The rate of encapsulation of the mRNA was measured using Quant-iTRiboGreen RNA Assay kit (Invitrogen Corp.) according to the attacheddocument.

Specifically, the mRNA in the nucleic acid lipid particle dispersion wasquantified in the presence and absence of a 0.015% Triton X-100detergent, and the rate of encapsulation was calculated according to thefollowing expression:{[Amount of the mRNA in the presence of the detergent]−[Amount of themRNA in the absence of the detergent]}/[Amount of the mRNA in thepresence of the detergent]}×100 (%)(3) Ratio of mRNA to Lipid

The amount of the phospholipid in the nucleic acid lipid particledispersion was measured using Phospholipid C-Test Wako (Wako PureChemical Industries Ltd.) according to the attached document.Specifically, the phospholipid in the sample was quantified in thepresence of a 1% Triton X-100 detergent.

The amounts of cholesterol and LP in the nucleic acid lipid particledispersion were measured by reverse-phase chromatography (system:Agilent 1100 series, column: Chromolith Performance RP-18 endcapped100-3 monolithic HPLC-column (Merck), buffer A: 0.01% trifluoroaceticacid, buffer B: 0.01% trifluoroacetic acid and methanol, gradient (B %):82-92% (0-10 min), flow rate: 2 mL/min, temperature: 50° C., detection:205 nm).

The total amount of lipids was calculated from the amount of thephospholipid, the amount of cholesterol, and the amount of LP, and thecompositional ratio of lipid components constituting the liposome, andthe ratio of the mRNA to the lipid was calculated from the “amount ofthe mRNA in the presence of the detergent” of the preceding paragraph(2) according to the following expression:[mRNA concentration in the presence of the detergent]/[Total lipidconcentration] (wt/wt)

The results are shown in Table 21.

TABLE 21 Ratio of mRNA to Rate of lipid encapsulation of mRNA/lipidAverage particle mRNA mRNA (%) (wt/wt) size (nm) mCherry 98 0.037 151 ±71 FLuc 98 0.038 145 ± 56

These results showed that the mRNA was encapsulated in the lipidparticle, and this nucleic acid lipid particle had an average particlesize of approximately 100 nm to approximately 200 nm.

Test Example 11

As described below, the expression of mCherry was measured using anucleic acid lipid particle prepared using a novel lipid.

(1) Transfection

The concentration of a human hepatocellular carcinoma HuH-7 cell linewas adjusted to 10,000 cells/mL in a DMEM medium (manufactured byInvitrogen Corp.) containing 10% fetal bovine serum (culture medium).Then, the resulting culture solution was inoculated at 100 μL/well to a96-well flat-bottomed plate (manufactured by Corning Inc./Falcon) andcultured at 37° C. for 1 day under 5.0% CO₂. The nucleic acid lipidparticle dispersion prepared in Example 58 was diluted with a culturemedium to prepare dilution series having final mRNA concentrations of 2,0.4, and 0.08 μg/mL in the medium. Then, each dilution was added to thecells after removal of the culture supernatant, and the culture wasfurther continued. This operation was performed at N=3 for eachconcentration. A control group was cultured in only a culture medium.

(2) Fluorescent Observation of mCherry

One day after the transfection, the culture medium was removed, and 100μL of 10 N Mildform (Wako Pure Chemical Industries Ltd.) was added toeach well and left at room temperature for 10 minutes in the dark. Afterwashing off of 10 N Mildform with DPBS (Dulbecco's PBS, LifeTechnologies, Inc.), 50 μL of Hoechst 33342, trihydrochloride,trihydrate (manufactured by Invitrogen Corp.) concentration-adjusted to20 μg/mL with DPBS was added to each well and left at room temperaturefor 1 hour in the dark. After replacement with DPBS, the fluorescencewas observed using IN Cell Analyzer 6000 (GE Healthcare Japan Corp.).The measurement conditions were as follows: Hoechst; excitationavelength: 405 nm, detection wavelength: 455 nm/50 nm (filter centralwavelength/band width), and mCherry; excitation wavelength: 561 nm,detection wavelength: 605 nm/52 nm (filter central wavelength/bandwidth).

The results are shown in FIG. 6. The upper boxes of this diagram depictimages of nuclei stained with Hoechst, and the lower boxes depict imagesof mCherry. The nucleic acid lipid particle having the compound ofExample 8 was found to promote the expression of mCherry. These resultsdemonstrated that the nucleic acid lipid particle containing thecompound of Example 8 is useful as a nucleic acid lipid particle capableof promoting the expression of mRNA.

(3) Measurement of Expression Level of Luciferase

Six hours after the transfection, the expression level of luciferase wasmeasured using Luciferase reporter Gene Assay, high sensitivity(manufactured by F. Hoffmann-La Roche, Ltd.) according to the attacheddocument. An average value of relative luminescent units (RLU) at N=3 isshown in Table 22.

TABLE 22 mRNA concentration (μg/mL) RLU (1 × 10⁵) 0 0 0.08 0.7 0.4 2.72.0 3.7

As a result, the nucleic acid lipid particle having the compound ofExample 8 was found to promote the expression of FLuc. These resultsdemonstrated that the nucleic acid lipid particle containing thecompound of Example 8 is useful as a nucleic acid lipid particle capableof promoting the expression of mRNA.

Example 60 Preparation of Double-stranded Polynucleotide-encapsulatedNucleic Acid Lipid Particle

Distearoylphosphatidylcholine(1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter, referred to asDSPC, NOF CORPORATION), cholesterol (hereinafter, referred to as Chol,Sigma-Aldrich, Inc.), the compound described in Example 8 (hereinafter,referred to as LP), and N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (hereinafter,referred to as PEG-C-DMA) were prepared at a molar ratio ofDSPC:Chol:LP:PEG-C-DMA=10:48:40:2 into a lipid solution having a totallipid concentration of 26.8 mM in ethanol.

The concentration of a double-stranded polynucleotide described inNature Biotechnology (2008) 26, 561-569 (siFVII: siRNA against mouseFactor VII) was adjusted to 1 mg/mL with a citrate buffer solution (10mM citrate buffer, pH 4.0) containing 30% ethanol to obtain adouble-stranded polynucleotide solution.

The lipid solution, the double-stranded polynucleotide solution, and acitrate buffer solution (20 mM citrate buffer, pH 4.0) were heated to37° C. The lipid solution was added dropwise to the citrate buffersolution (20 mM citrate buffer, pH 4.0) and mixed therewith such thatthe volume ratio between the lipid solution and the citrate buffersolution was 3:7 to obtain a crude liposome dispersion. Subsequently,the crude liposome dispersion was added dropwise to the double-strandedpolynucleotide solution and mixed therewith such that the ratio (N/P) ofthe number of LP molecules (N) to the number of double-strandedpolynucleotide-derived phosphorus atoms (P) was a molar ratio describedin Table 23. The mixture was incubated at 37° C. for 30 minutes toobtain a nucleic acid lipid particle dispersion. The nucleic acid lipidparticle dispersion was dialyzed against approximately 100 mL of aphosphate buffer solution (pH 7.4) for 12 to 18 hours (Float-A-Lyzer G2,MWCO: 100 kD, Spectra/Por) for the removal of ethanol and the removal ofunencapsulated double-stranded polynucleotides by neutralization toobtain a purified dispersion of a nucleic acid lipid particle containingthe double-stranded polynucleotide and the lipid described in Example 8.

TABLE 23 N/P ratio Particle 28 2.0 Particle 29 2.5 Particle 30 3.0Particle 31 3.5 Particle 32 4.0 Particle 33 4.5 Particle 34 5.0 Particle35 6.0

Example 61 Characterization of Double-strandedPolynucleotide-encapsulated Nucleic Acid Lipid Particle

The nucleic acid lipid particle dispersion prepared in Example 60 wascharacterized. The characterization was conducted by the methodsdescribed in Example 32, and the rate of polynucleotide encapsulation inthe nucleic acid lipid particle described in Example 60, the weightratio of the polynucleotide to the lipid, and the average particle sizeare shown in Table 24.

TABLE 24 Rate of encapsulation siRNA/lipid Particle size N/P ratio* (%)(wt/wt)** (nm) Particle 28 2.0 96 0.128 126 ± 40 Particle 29 2.5 970.099 134 ± 9 Particle 30 3.0 97 0.085 139 ± 50 Particle 31 3.5 98 0.066147 ± 39 Particle 32 4.0 98 0.063 142 ± 43 Particle 33 4.5 98 0.057 141± 23 Particle 34 5.0 99 0.051 137 ± 26 Particle 35 6.0 99 0.037 151 ± 38*N/P ratio: ratio of the number of molecules of LP (N) to the number ofphosphorus atoms derived from double-stranded polynucleotide (P)**siRNA/lipid (wt/wt): weight ratio of polynucleotide to lipid

These results showed that the double-stranded polynucleotide wasencapsulated in the lipid particle, and this nucleic acid lipid particlehad an average particle size of approximately 100 nm to approximately200 nm.

Test Example 12 Factor VII (FVII) Protein Measurement

The Factor VII protein was measured according to a method described inNature Biotechnology (2010) 28, 172-176. C57BL6/J mice (male, 9 weeksold) were randomly grouped (n=4). The nucleic acid lipid particledispersion prepared in Example 60 was intravenously injected at a doseof 0.3 mg/kg to the tail of each mouse. One day after theadministration, approximately 50 μL of blood was collected from the tailvein, and plasma was obtained. The amount of the Factor VII protein inthe obtained plasma was measured using Biophen FVII assay kit(manufactured by Aniara Corp.) according to the attached protocol.

When the amount of FVII of respective plasma samples collected in equalamounts from individuals in a PBS administration group was defined as100%, the relative ratio (%) of the amount of FVII in a plasma sample ofeach individual was used as a measurement value (A). An average value(B) was determined from the respective measurement values of theindividuals in the PBS administration group. The relative ratio of themeasurement value (A) of each individual was determined from theexpression: A/B×100 (%). The average value of the relative ratios in theadministration group of each nucleic acid lipid particle is shown inTable 25. As a result, as shown in Table 25, particles 28 to 35 as thenucleic acid lipid particles prepared in Example 60 exhibited strongFVII inhibitory activity. These results demonstrated that a nucleic acidlipid particle having lipid composition as found in particles 28 to 35is useful as a nucleic acid lipid particle capable of inhibiting geneexpression.

TABLE 25 Relative amount of FVII (%) PBS 100 Particle 28 38 Particle 2911 Particle 30 <10 Particle 31 <10 Particle 32 31 Particle 33 <10Particle 34 <10 Particle 35 <10

INDUSTRIAL APPLICABILITY

The present invention may provide a novel cationic lipid that forms alipid particle in combination with an amphipathic lipid, a sterol, and alipid reducing aggregation during lipid particle formation.

The present invention may also provide a lipid particle comprising thecationic lipid.

The present invention may further provide a nucleic acid lipid particlecomprising the lipid particle and further a nucleic acid. The nucleicacid lipid particle of the present invention can be used in apharmaceutical composition.

Free Text of Sequence Listing

-   SEQ ID NO: 1: CT-169-   SEQ ID NO: 2: CT-157-   SEQ ID NO: 3: CT-103-   SEQ ID NO: 4: CT-292-   SEQ ID NO: 5: CT-315-   SEQ ID NO: 6: CT-387-   SEQ ID NO: 7: Sense strand region of CT-454-   SEQ ID NO: 8: Antisense strand region of CT-454-   SEQ ID NO: 9: Sense strand region of HS-005-   SEQ ID NO: 10: Antisense strand region of HS-005-   SEQ ID NO: 11: Sense strand region of HS-006-   SEQ ID NO: 12: Antisense strand region of HS-006-   SEQ ID NO: 13: Sense strand region of HS-005s-   SEQ ID NO: 14: Antisense strand region of HS-005s-   SEQ ID NO: 15: Sense strand region of HS-006s-   SEQ ID NO: 16: Antisense strand region of HS-006s

The invention claimed is:
 1. A lipid of formula (Ia):

or a pharmacologically acceptable salt thereof, wherein R¹ and R² areeach a methyl group; R⁸ is a hydrogen atom; L¹ is a C₁₇-C₁₉ alkenylgroup optionally substituted with a C₂-C₄ alkanoyloxy group; L² is aC₁₀-C₁₂ alkyl group or a C₁₀-C₁₂ alkenyl group; m is 0; p is 0, 1, or 2;and r is 0, 1, 2, or 3, provided that p+r is 2 or
 3. 2. The lipid ofclaim 1 or a pharmacologically acceptable salt thereof, wherein L¹ is aC₁₇-C₁₉ alkenyl group optionally substituted with an acetyloxy group. 3.The lipid of claim 1 or a pharmacologically acceptable salt thereof,wherein L¹ is a heptadecenyl group, an octadecenyl group, a nonadecenylgroup, a heptadecadienyl group, an octadecadienyl group, anonadecadienyl group, a heptadecatrienyl group, an octadecatrienylgroup, or a nonadecatrienyl group optionally substituted with anacetyloxy group.
 4. The lipid of claim 1 or a pharmacologicallyacceptable salt thereof, wherein L¹ is a(R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-9-octadecenyl group(oleyl group), a cis-8,11-heptadecadienyl group, acis-9,12-octadecadienyl group (linoleyl group), acis-10,13-nonadecadienyl group, or a cis-6,9,12-octadecatrienyl group(linolenyl group).
 5. The lipid of claim 1 or a pharmacologicallyacceptable salt thereof, wherein L² is a C₁₀-C₁₂ alkyl group.
 6. Thelipid of claim 1 or a pharmacologically acceptable salt thereof, whereinL² is a decyl group, a decenyl group, an undecyl group, an undecenylgroup, a dodecyl group, a dodecenyl group, a decadienyl group, anundecadienyl group, or a dodecadienyl group.
 7. The lipid of claim 1 ora pharmacologically acceptable salt thereof, wherein L² is a decylgroup, or a cis-7-decenyl group.
 8. The lipid of claim 1 or apharmacologically acceptable salt thereof, wherein p+r is
 2. 9. Thelipid of claim 1 or a pharmacologically acceptable salt thereof, whereinthe lipid has the formula:


10. The lipid of claim 1 or a pharmacologically acceptable salt thereof,wherein the lipid has the formula:


11. The lipid of claim 1 or a pharmacologically acceptable salt thereof,wherein the lipid has the formula:


12. The lipid of claim 1 or a pharmacologically acceptable salt thereof,wherein the lipid has the formula:


13. The lipid of claim 1 or a pharmacologically acceptable salt thereof,wherein the lipid has the formula:


14. A lipid particle comprising the lipid of claim
 1. 15. The lipidparticle of claim 14, further comprising a lipid capable of reducingaggregation during lipid particle formation.
 16. The lipid particle ofclaim 15, wherein the lipid capable of reducing aggregation during lipidparticle formation is a PEG-lipid.
 17. The lipid particle of claim 16,wherein the PEG-lipid is N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA), or1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol.
 18. The lipidparticle of claim 16, wherein the PEG-lipid is N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA). 19.The lipid particle of claim 16, wherein the PEG-lipid is N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dipalmityloxypropyl-3-amine(PEG-C-DPA), or 1,2-dipalmitoyl-sn-glycerol methoxypolyethylene glycol.20. The lipid particle of claim 16, wherein the PEG-lipid is N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dipalmityloxypropyl-3-amine(PEG-C-DPA).
 21. The lipid particle of claim 16, wherein the PEG-lipidis N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearyloxypropyl-3-amine (PEG-C-DSA), or1,2-distearoyl-sn-glycerol methoxypolyethylene glycol.
 22. The lipidparticle of claim 16, wherein the PEG-lipid is N-[methoxy poly(ethyleneglycol)2000]carbamoyl]-1,2-distearyloxypropyl-3-amine (PEG-C-DSA). 23.The lipid particle of claim 16, wherein the PEG-lipid has a molecularweight of 1,000 to 5,000.
 24. The lipid particle of claim 16, whereinthe PEG-lipid has a molecular weight of 1,800 to 2,200.
 25. The lipidparticle of claim 14, further comprising a sterol.
 26. The lipidparticle of claim 25, wherein the sterol is cholesterol.
 27. The lipidparticle of claim 25, further comprising an amphipathic lipid.
 28. Thelipid particle of claim 27, wherein the amphipathic lipid is selectedfrom distearoylphosphatidylcholine (DSPC),dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine(DMPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC),dioleoylphosphatidylethanolamine (DOPE), and sphingomyelin (SM).
 29. Thelipid particle of claim 27, wherein the amphipathic lipid isdistearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine(DPPC).
 30. The lipid particle of claim 27, wherein the lipidcomposition of the amphipathic lipid, the sterol, the lipid, and thelipid capable of reducing aggregation during lipid particle formation is25% or less of the amphipathic lipid, 15% or more of the sterol, 20% to70% of the lipid, and 1% to 10% of the lipid capable of reducingaggregation during lipid particle formation, in terms of molar quantity.31. The lipid particle of claim 27, wherein the lipid composition of theamphipathic lipid, the sterol, the lipid, and the lipid capable ofreducing aggregation during lipid particle formation is 15% or less ofthe amphipathic lipid, 32% or more of the sterol, 45% to 65% of thelipid, and 1.5% to 3% of the lipid capable of reducing aggregationduring lipid particle formation, in terms of molar quantity.
 32. A lipidparticle comprising the lipid particle of claim 14 and a nucleic acid.33. The lipid particle of claim 32, wherein the nucleic acid is selectedfrom the group consisting of a single-stranded DNA, a single-strandedRNA, a single-stranded polynucleotide of a DNA and an RNA mixed witheach other, a double-stranded DNA, a double-stranded RNA, a DNA-RNAhybrid polynucleotide, and two polynucleotides of a DNA and an RNA mixedwith each other.
 34. The lipid particle of claim 32, wherein the nucleicacid is a single-stranded or double-stranded polynucleotide having anRNA interference effect.
 35. The lipid particle of claim 32, wherein thenucleic acid is a single-stranded RNA.
 36. The lipid particle of claim32, wherein the ratio of the number of molecules of the lipid to thenumber of phosphorus atoms derived from the nucleic acid is 2.0 to 9.0.37. The lipid particle of claim 32, wherein the ratio of the number ofmolecules of the lipid to the number of phosphorus atoms derived fromthe nucleic acid is 3.0 to 9.0.
 38. The lipid particle of claim 32,wherein the average particle size is approximately 30 nm toapproximately 300 nm.
 39. The lipid particle of claim 32, wherein theaverage particle size is approximately 30 nm to approximately 200 nm.40. The lipid particle of claim 32, wherein the average particle size isapproximately 30 nm to approximately 100 nm.
 41. A pharmaceuticalcomposition comprising the nucleic acid lipid particle of claim 32 and apharmaceutically acceptable carrier.
 42. A method of inhibiting theexpression of a target gene, comprising administering the nucleic acidlipid particle of claim 32 to a mammal, wherein the nucleic acid lipidparticle has an RNA interference effect and/or a gene inhibitory effecton said target gene.
 43. A method of treating a disease derived from theexpression of a target gene, comprising administering the nucleic acidlipid particle of claim 32 to a mammal, wherein the nucleic acid lipidparticle has a gene expression effect on the target gene.
 44. The methodof claim 43, wherein the disease derived from the expression of a targetgene is cancer.
 45. The method of claim 43, wherein the disease derivedfrom the expression of a target gene is cancer, liver disease,gallbladder disease, fibrosis, anemia, or genetic disease.